Adjunct Professor, Senior Researcher Ari Väänänen
email: ari.vaananen@ttl.fi

Dr Kirsi Ahola

M.Sc Mr Aki Koskinen

M.Soc.Sc Ms Krista Pahkin

Dr Anne Kouvonen

Organizational Merger and Psychiatric Morbidity: A Prospective Study in a Changing Work Organization

Keywords: Psychiatric disorders, Prospective studies, Organizational change

Background: Prospective studies on the relationship between organizational merger and mental health have been conducted using subjective health indicators. The objective of this prospective occupational cohort study was to examine whether a negative change during an organizational merger is an independent predictive factor of psychiatric morbidity.

Method

: Survey data on organizational characteristics, health, and other factors were collected prior to (1996) and after the merger (2000); register data on psychiatric morbidity at baseline (1/1994-9/2000) and during the follow-up (10/2000-12/2005). Participants were 6 511 (77% men) industrial employees aged 21-65 with no register-based diagnosed psychiatric events prior to the follow-up (the Still Working Study). During the follow-up, 252 participants were admitted to hospital due to psychiatric disorders, were prescribed a psychotropic drug, or attempted or committed suicide.

Results: A negative self-reported change in the work organization during the merger was associated with increased risk of post-merger psychiatric event (Hazard Ratio 1.60; 95% Confidence Interval 1.19-2.14). This association was independent of mental health-related factors measured before the merger announcement, such as demographic characteristics, occupational status, personal orientation to life, self-rated health, self-reported psychiatric morbidity, or chronic disease.

Conclusions:

A negative change in work organization during an organizational merger may elevate the risk for post-merger psychiatric morbidity.

Introduction

To stay competitive and efficient in the current global business environment, companies often have to merge with one another. Mergers often cause considerable changes in work organizations, work teams, roles, and job statuses. From the employees' perspective, factors such as continuity [START_REF] Van Knippenberg | Organizational identification after a merger: A social identity perspective[END_REF], security [START_REF] Bordia | Uncertainty during organizational change: types, consequences, and management strategies[END_REF], and predictability

(3) may be threatened. Merger-related changes may therefore worsen working conditions [START_REF] Gulliver | Staff morale in the merger of mental health and social care organizations in England[END_REF][START_REF] Osthus | For better or worse? Workplace changes and the health and well-being of Norwegian workers[END_REF][START_REF] Schweiger | Human resources: The forgotten factor in mergers and acquisitions[END_REF], increase psychopathological symptoms [START_REF] Ferrie | The health effects of major organizational change and job insecurity[END_REF], and have adverse effects on employees' mental health [START_REF] Ferrie | The health effects of major organizational change and job insecurity[END_REF][START_REF] Hansson | Organizational change, health, and sick leave among health care employees. A longitudinal study measuring stress markers, individual, and work site factors[END_REF][START_REF] Michie | Reducing work-related psychological ill health and sickness absence: a systematic literature review[END_REF].

Although organizational mergers have become globally increasingly common, surprisingly little is known on the mental health effects related to them. Longitudinal studies on the health effects of organizational changes have mainly dealt with the effects of downsizing [START_REF] Vahtera | Organizational downsizing, sickness absence, and mortality: 10-town prospective cohort study[END_REF], changes in labour market status [START_REF] Martikainen | The effects of unemployment on mortality following workplace downsizing and workplace closure: a register-based followup study of Finnish men and women during economic boom and recession[END_REF], employment instability [START_REF] Virtanen | Temporary employment and health[END_REF], and reorganizations within one workplace [START_REF] Ferrie | The health effects of major organizational change and job insecurity[END_REF]. Both the survivors of the downsized firms and workers with an unstable job suffer from mental health problems (e.g., increased use of psychotropic drugs) more often than those who have not experienced downsizing or who have a more stable job [START_REF] Ferrie | The health effects of major organizational change and job insecurity[END_REF] [START_REF] Virtanen | Temporary employment and health[END_REF] [START_REF] Kivimäki | Organizational downsizing and increased use of psychotropic drugs among employees who remain in employment[END_REF]. Indeed, sparse prospective evidence suggests that a decline in one's status during a merger may be associated with at least a temporary decline in one's psychological well-being [START_REF] Väänänen | Maintenance of subjective health during a merger: The role of experienced change and pre-merger social support at work in white-and blue-collar workers[END_REF].

However, no long-term prospective studies have been conducted on the impact of an organizational merger on the post-merger psychiatric morbidity of employees.

In this five-year prospective cohort study, our objective was to examine whether a self-assessed change (improved or worsened situation) during an organizational merger (8/1998-9/2000) predicted the onset of new psychiatric events (measured by hospitalization, drug prescription, attempted or committed suicide) after it (10/2000-12/2005) among employees who did not have a psychiatric disorder prior to or during the merger according to the health records (1/1994-9/2000). In addition, we examined whether the association was independent of various indicators of pre-merger health status and other baseline characteristics, and whether certain socio-demographic groups were particularly vulnerable to the negative effects of a merger.

Methods

Procedure

Data were derived from the ongoing Still Working prospective cohort study examining the work-related antecedents of morbidity and mortality in a multinational forest industry corporation [START_REF] Kouvonen | Sense of coherence and psychiatric morbidity. A 19-year register-based prospective study[END_REF][START_REF] Väänänen | Lack of predictability at work and risk of acute myocardial infarction: An 18-year prospective study of industrial employees[END_REF]. In this study, we used a link between a personal identification code of the surveys and the national ID number given to all Finns at birth, to merge the questionnaire data with data from national health registers. The approval of the ethics committee of the Finnish Institute of Occupational Health was obtained for the study.

Study context

The merger was performed between two companies based originally in Finland and Sweden employing more than 40 000 employees altogether globally. It was announced in the summer of 1998 and was carried out in 1999 and in early 2000. In Finland, where this prospective study was conducted, the company had been a stable traditional employer for several decades. The majority of the industrial plants were dispersed around the country, often being the major employer in the sparsely populated local areas. The merger did not lead to dramatic changes such as large-scale redundancies during the study period, but evident changes in work roles, supervision, information practices, and other procedures did take place.

Study design

As Figures 1 and2 show, we collected the data in four phases, twice before the merger and twice after. At Time 1, the data on baseline psychiatric morbidity were collected between January 1, 1994 and September 30, 2000 from the national health registers (Hospital Discharge Register, Drug Imbursement Register). In spring 1996, prior to the merger, data on the subpopulation of employees who responded to the survey on psychosocial factors and subjective health were gathered (Time 2). During autumn 2000, a post-merger survey (Time 3) on work conditions, recent organizational changes, and subjective health was carried out. In both survey phases, the questionnaires were sent to the work units, distributed to employees by their supervisors, and, once completed, mailed directly to the Finnish Institute of Occupational Health. Participation was voluntary, and confidentiality was assured to all employees. After the second survey assessment, data on post-merger psychiatric morbidity from October 1, 2000 to December 31, 2005 (Time 4) was collected from the national mortality register and the same national health registers as at Time 1.

Participants

Ascertainment of Psychiatric Events

Data on all persons who had been hospitalized for psychiatric disorders (ICD9 codes: 291-319; ICD10 F04-F99), who had been prescribed a psychotropic drug (ATC codes N05A, N05B, N06A) or had attempted suicide before the assessment of organizational change (1/1994-9/2000) and after (10/2000-12/2005), were obtained from the Hospital Discharge Register and from the Drug Imbursement Register, respectively. These registers reliably cover all information on the hospital admissions and drug prescription purchases for each Finnish citizen residing in Finland.

Diagnosis-specific data related to depression were also used (ICD9 296.1, 300.4A; ICD10 F32, F33, D34.1; ATC N06A). In addition, data on suicides (10/2000-12/2005) from the Statistics Finland national mortality register was collected.

Ascertainment of mortality

The dates and causes of death from 1 October 2000 to 31 December 2005 were obtained from Statistics Finland. The database provides virtually complete population mortality data.

Assessment of Demographic, Health-related, and Psychosocial Baseline

Characteristics

In Study Population I, data on covariates were collected at Time 1 (pre-merger psychiatric events) and at Time 3 (socio-demographic characteristics). All data on socio-demographic characteristics were obtained from the National Population Register Centre, except occupational status (employer's records). Marital status (married versus unmarried) and occupational status (manual versus non-manual employees) were used as dichotomized variables. Age was used as a continuous measure, except in the age-stratified analyses (under 50 versus 50 or over). In Study Population II, job characteristics were assessed at Time 2 using the Occupational Stress Questionnaire [START_REF] Elo | Occupational stress questionnaire: User's instructions[END_REF], and sense of coherence (α= 0.86) -a health-enhancing personality characteristic -was measured using a 13-item version of Antonovsky's Orientation to Life Questionnaire [START_REF] Kouvonen | Sense of coherence and psychiatric morbidity. A 19-year register-based prospective study[END_REF][START_REF] Antonovsky | The structure and properties of the sense of coherence scale[END_REF]. Both decision authority (α= 0.80) and skill discretion (α= 0.83) at work were measured using five items [START_REF] Väänänen | Lack of predictability at work and risk of acute myocardial infarction: An 18-year prospective study of industrial employees[END_REF][START_REF] Väänänen | Job characteristics, symptoms of strain and social support as antecedents of sickness absences in men and women in the private industrial sector[END_REF]. Summary scales were computed. For descriptive purposes, the scales were trichotomised, while in the main analyses they were used as continuous measures. At Time 2, data on pre-merger subjective health status was also collected using measures of self-reported psychiatric morbidity (10-item measure on anxiety, depression, and other symptoms ( 14)), selfrated health (1-item measure [START_REF] Idler | Self-rated health and mortality: A review of twenty-seven community studies[END_REF], average/good versus bad), and chronic diseases (no versus yes). Self-reported psychiatric morbidity was used as a continuous variable while other health measures were used as dichotomized variables in the analyses.

Statistical Analysis

Descriptive statistics were applied to obtain the prevalence of organizational change during a merger and the prevalence of psychiatric events by covariates. The associations between organizational change levels and confounding factors were described by cross-tabulations and χ2-tests. The association between organizational change and psychiatric morbidity was assessed using Cox proportional hazards models. For each participant, person-days of follow-up were calculated from October 1, 2000 to the death of the employee, his or her hospitalization or drug purchase or death due to psychiatric reasons, or to December 31, 2005, whichever of these three options came first. The time-dependent interaction term between predictor and logarithm of the follow-up period was not statistically significant, confirming that the proportional hazards assumption was justified (all p values > 0.32).The adjusted hazard ratios (HRs) and 95% confidence intervals (95% CIs) for three categories of organizational change (improved, unaltered, or worsened situation) were calculated, and the unaltered situation was used as a reference category. The main analyses were conducted in four steps. First, age and sex were adjusted for. Second, marital status and occupational status were also adjusted for. Third, sense of coherence and job characteristics were added to the models in the Study Population II, information of which could be used from Time 2. Fourth, the subjective health measures from Time 2 were additionally adjusted for in the same population. Additional analyses were conducted separately for other than alcohol and drug-related diagnoses and for diagnoses related to depression. In the Study Population II, stratified analyses were run by socio-demographic characteristics. The analyses were conducted using the PHREG procedure of the SAS 9.2 statistical software package.

Results

Of all 6 511 participants with neither hospital admissions for psychiatric disorders nor use of psychotropic drugs at baseline, 59 died during the follow-up and were censored at the time of death. By the end of the follow-up, a total of 44 participants were admitted to hospital due to a psychiatric disorder, 199 were prescribed psychotropic medication by a physician, 5 participants committed and 4 attempted suicide (total n of cases=252, 3.9% of the study population). The mean time before the first event was 2.7 years (range 0.0-5.2 years). As Table 1 shows, employees with a psychiatric disorder were more often older employees and men. Employees with a psychiatric event after the merger had reported more symptoms of psychiatric morbidity, poorer self-rated health, chronic disease, and a lower sense of coherence prior to the merger than employees with no psychiatric events. Table 2 presents variations in the prevalence of organizational change by demographic and other factors. Nearly 60% of the participants had experienced the organizational change rather neutrally, more than 20% saw that the situation had improved, while nearly 20% considered the changes during the merger negative. The participants who were over 50, had low self-rated health and a chronic disease prior to the merger, reported pre-merger symptoms of psychiatric morbidity, had a weak sense of coherence and weak psychosocial work characteristics, reported negative organizational changes more often than the others. Non-manual employees experienced both negative and positive changes more frequently than manual employees.

As Table 3 shows, after adjustment for age, sex, marital status, and occupational status, a negative change in the organization during the merger was associated with a 60% increased risk of psychiatric events (95% CI=1. 19-2.14). The association slightly attenuated but remained statistically significant in the Study Population II, even after the work-related psychosocial risk factors, sense of coherence, and various dimensions of subjective health status measured prior to the merger announcement were controlled (HR=1.53, 95% CI=1.06-1.99). The associations were more or less identical after alcohol-related psychiatric disorders (n=31) were excluded. The negative change in the organization during the merger was also related to subsequent events of depression. In all models, the participants who reported a negative change in organization showed the shortest time before the onset of psychiatric event.

Table 4 shows rather similar associations between organizational change and psychiatric events in separate groups according to age and sex. However, non-manual employees who had experienced the negative organizational change had a 2-fold risk of subsequent psychiatric events (95% CI=1.25-3.18) whereas manual employees were not significantly affected by organizational changes (HR=1.38, 95% CI=0.94-2.03).

Discussion

Our unique longitudinal data connecting organizational surveys and national health registers and covering a period of 11 years with four assessment phases reveal that merger-related experiences may affect employees' mental health long after the merger process is over. Evidence suggests that when initially healthy employees considered the situation after an organizational merger worse than before, it predicted the onset of a psychiatric event during the five-year follow-up period after the merger. The association was independent of conventional mental health risk factors. The finding lends support to the earlier longitudinal findings on organizational changes and mental health using both register-based ( 12) and subjective (6, 13) measures of psychiatric morbidity as outcomes.

A merger can change one's work in many ways, for example the nature of the job itself, the human relations in one's work team, and the information flow in the organization [START_REF] Cartwright | Managing workplace stress[END_REF]. A merger may increase demands at work (4) and result in sleep disturbances [START_REF] Ferrie | The health effects of major organizational change and job insecurity[END_REF]. A negative organizational change is likely to be experienced as a potential threat which can exceed the adaptive resources of the employee (21) via various pathways such as uncertainty (2), decreased status (13), role conflicts [START_REF] Gulliver | Staff morale in the merger of mental health and social care organizations in England[END_REF][START_REF] Schweiger | Human resources: The forgotten factor in mergers and acquisitions[END_REF], and anxiety [START_REF] Buono | The human side of mergers and acquisitions: Managing collision between people, cultures and organizations[END_REF]. Based on our data, it seems plausible that these types of mergerrelated stressful negative experiences can lead to elevated risk of psychiatric events after the most hectic period of organizational change has been passed.

Previous evidence on the association between organizational merger and mental health problems has mainly been based on subjective outcomes, and the analyses have not sufficiently controlled for the confounding effect of permanent personality characteristics, job content, or baseline mental health status. Thus, chronically weak psychosocial resources and/or personal orientation to life may have confounded the associations [START_REF] De Lange | Different mechanisms to explain the reversed effects of mental health on work characteristics[END_REF]. Pre-existing mental or chronic illness may also have a role in the organizational change-psychiatric morbidity relationship. We believe that this study is able to surmount these weaknesses. First, we used register data to assess the outcome. Furthermore, all those who had had psychiatric problems according to the medical records prior to the assessment of merger were excluded from the cohort. Finally, in the additional analyses (Study Population II), the pre-merger subjective measures of psychiatric morbidity, general health status, personal orientation to life, and job content were used as covariates. Therefore, due to our exclusion criteria and the large set of covariates used, health selection, personal orientation to life, or adverse job content were unlikely to play a major role in our results.

It is noteworthy that our stratified analyses by occupational status showed that, unlike the manual employees, the non-manual employees who were often responsible for carrying out changes during the merger, were at a particularly high risk of psychiatric morbidity after the merger period if they had reacted negatively to the organizational changes. Some weaknesses may limit the generalizability of our findings. It is possible that some unexplored factors such as severe negative life events may have contributed to mental health risk and confounded the associations. However, it is likely that personal life crises have a rather independent or additional role in the impairment of mental health with regard to organizational changes. A great proportion of our study participants worked at industrial plants in rather small and often remote communities, where it is difficult to find a new job.

Hence, for those employees who had experienced negative changes and with family responsibilities, future attempts to find a new job would have required major changes in their lives. This may have increased the impact of the negative organizational changes. In contrast, lengthy follow-up after the merger and exclusion of employees with psychiatric events during the merger (1999)(2000) attenuated the relationship between the stressor and the outcome, and this is likely to have minimized the estimated associations between organizational change and psychiatric morbidity. Yet we acknowledge that the findings of the present study should be validated in further large-scale studies. Such a replication would be useful in determining the generalizability of our findings, i.e., whether they apply to different types of merging organizations; and whether differences between societies and sectors of work moderate the effects of an organizational merger on employee psychiatric morbidity.

Practical implications

Organizational mergers and other organizational changes have become increasingly common throughout the world. The steep increase in new cases of mental ill health among working population may partly reflect the incapacity to manage organizational changes properly [START_REF] Cherry | Reported incidence and precipitating factors of work-related stress and mental ill-health in the United Kingdom (1996-2001)[END_REF]. Our longitudinal data suggest that negative experiences during a merger may lead to elevated psychiatric morbidity among employees with no earlier register-based diagnosed psychiatric disorders, especially non-manual employees seem to be vulnerable.

Policymakers, employers, and occupational health professionals should recognize that an organizational merger may pose a severe risk to employees' mental health. Organizational changes such as mergers may, therefore, be important to consider in mental health prevention of employed population. Procedures for managing the inevitable organizational changes in a way which would minimize negative experiences should be further studied and implemented.

  Two study populations were formed. When generating Study Population I (including assessments at Time 1, 3, and 4), we excluded participants with a history of hospital admissions or drug purchases related to psychiatric disorders by baseline (n=420) from the eligible study population (n=9 705). The final cohort of 6 511 employees consisted of employees who (1) were free from psychiatric disorders (no recorded hospital admissions or drug purchases) before the survey on October 1, 2000; (2) had worked for the company for at least 12 months before the survey in 2000; (3) responded to the scale of organizational change; (4) had no missing values for any other study variables; and (5) were identified from the database of the National Population Register Centre. The mean length of follow-up was five years and two months (range 0.0-5.3 years). At Time 3, the mean age was 45 (range 21-65). Women (23% versus 22%, p=0.031), non-manual workers (36% versus 28%, p<0.001), and married participants (63% versus 61%, p=0.001) were overrepresented in the final study population whereas no difference emerged according to age. The same exclusion criteria (1-5) was applied in Study Population II of 4 096 employees who had responded to the surveys both before (Time 2) and after the merger (Time 3). Their baseline characteristics were more or less identical to the Study Population I; the participants were only somewhat older (mean age 47). The use of this population offered an opportunity to control the impact of several potential confounders measured prior to the merger announcement. Assessment of Change during Organizational Merger At Time 3 we assessed the employees' experiences of the merger period by asking what the course of events has been like during the organizational change. The study participants evaluated (1) the change in one's own standing at work (improved, unaltered, deteriorated), (2) the development within the work unit (1=very positive... very negative=5), and (3) the trends in the whole company (1=very positive... very negative=5) during the preceding one to two years. A summary measure of organizational change during a merger was derived by summing the responses across the three items (α =0.68). Based on summary scores, we formed three categories indicating the experienced change: improved situation (range 0-2.32), unaltered situation (range 2.33-3.32), and worsened situation (range 3.33-5).

Figure 1 Figure 2

 12 Figure 1 Study design, sample selection, and description of the Study Population I. Still

Table 1 Cox Proportional Hazard Ratios (HR) and Their 95% Confidence Intervals (95% CI) for New Psychiatric Events by Conventional Risk Factors in the Still Working Study a

 1

	Predictors	N	No of Cases HR (95% CI)	p-value
	Demographic characteristics (Time 3, N=6511)
	Age				0.001
	<50	4237	141	1.00
	50 or more	2274	111	1.03 (1.01 to 1.04)
	Sex				<0.001
	female	1490	84	1.00
	male	5021	168	1.67 (1.29 to 2172)
	Occupational status				0.077
	non-manual	2457	92	1.00
	manual	4054	160	1.34 (0.97 to 1.85)
	Marital status				0.236
	non-married	2265	92
	married	4246	160	0.84 (0.64 to 1.12)
	Health measures (Time 2, n=4096)
	Self rated health				0.015
	good or average	3937	151	1.00
	bad	159	12	2.08 (1.15 to 3.74)
	Chronic disease				0.002
	no	3201	109	1.00
	yes	895	54	1.70 (1.22 to 2.37)
	Self-reported psychiatric morbidity				<0.001
	low	3215	106	1.00
	high	881	57	1.85 (1.34 to 2.56)
	Psychosocial measures (Time 2, N=4096)
	Skill discretion at work				0.177
	high	1362	41	1.00
	intermediate	1515	65	1.37 (0.92 to 2.03)
	low	1219	57	1.44 (0.96 to 2.16)
	Decision authority at work				0.340
	high	1467	52	1.00
	intermediate	1390	63	1.31 (0.91 to 1.90)

a Adjusted for age and sex

Table 2 Experienced Organizational Change during the Merger by Demographic, Health-related, and Psychosocial Characteristics in the Still Working Study

 2

	Situation during the Merger

Acknowledgements

The research group was financially supported by the Finnish Work Environment Fund (OSH-ERA project, #109395). Dr Väänänen was also supported by the Academy of Finland (#128089). The authors thank all study participants, the Still Working members, and the leader of the earlier survey phases Raija Kalimo.

Funding

The research group was financially supported by the Finnish Work Environment Fund (OSH-ERA project, #109395). Dr Väänänen was also supported by the Academy of Finland (#128089).

What is already known on this subject?

Mergers often cause considerable changes in work organizations, work teams, roles, and job statuses. Negatively experienced merger-related changes seem to increase stress at work and have adverse effects on employees' subjective health.

What does this study add?

The results from this follow-up study covering a period of 11 years with four assessment phases indicate that merger-related stressful negative experiences can lead to elevated risk of psychiatric events. It seems that the negative change in the organization during the merger may represent a risk factor for psychiatric disorders.

Competing interests

None declared


R G Pleijhuis

G C Langhout

W Helfrich

G Themelis

A Sarantopoulos

L M A Crane

N J Harlaar

J S De Jong

V Ntziachristos

MD Gooitzen M Van Dam
email: g.m.van.dam@chir.umcg.nl

Near-Infrared Fluorescence (NIRF) Imaging in Breast-Conserving Surgery: Assessing Intraoperative Techniques in Tissue-Simulating Breast Phantoms

Keywords: Human breast cancer, near-infrared fluorescence, tissue-simulating phantoms, intraoperative imaging NIRF imaging in tissue-simulating breast phantoms

Near-infrared fluorescence (NIRF)-based tumour imaging has great potential for improving breastconserving surgery. In this report, intraoperative NIRF applications were pre-clinically evaluated using tissue-simulating breast phantoms equipped with fluorescent tumour-like inclusions of different size and shape in breast-conserving surgery simulations.

Introduction

Breast cancer is the most frequent malignancy in women worldwide with an estimated 1.4 million new cases in 2010 [START_REF] Parkin | Global Cancer Statistics[END_REF]. Breast-conserving therapy (BCT), consisting of breast-conserving surgery (BCS) followed by radiation therapy, has become the standard treatment for T1-T2 breast tumours and is generally regarded as sufficient for this subset of patients [START_REF] Schwartz | Consensus Conference on Breast Conservation[END_REF]. Unfortunately, a majority of studies on the surgical margin status after BCS have shown that positive margins are detected in 20 to 40% of patients, necessitating additional surgical intervention or radiotherapy [START_REF] Pleijhuis | Obtaining Adequate Surgical Margins in Breast-Conserving Therapy for Patients With Early-Stage Breast Cancer: Current Modalities and Future Directions[END_REF].

Two major points for improving outcome after BCS involve i) a more reliable intraoperative tumour localization and ii) improved real-time feedback on the presence of possible residual disease during or after excision of the tumour [START_REF] Singletary | Breast Cancer Surgery for the 21st Century: the Continuing Evolution of Minimally Invasive Treatments[END_REF].

Intraoperative application of an optical imaging technique known as near-infrared fluorescence (NIRF) imaging may improve the clinical outcome of BCS (3;5).

Near-infrared fluorescence imaging

In recent years, significant progress has been made in the development of optical imaging systems and fluorophores for clinical applications (6;7). Several animal (5;8-10) and clinical [START_REF] Tagaya | Intraoperative Identification of Sentinel Lymph Nodes by Near-Infrared Fluorescence Imaging in Patients With Breast Cancer[END_REF][START_REF] Sevick-Muraca | Imaging of Lymph Flow in Breast Cancer Patients After Microdose Administration of a Near-Infrared Fluorophore: Feasibility Study[END_REF][START_REF] Brandt | Randomized Control Trial of Fluorescence-Guided Surgical Excision of Nonmelanotic Cutaneous Malignancies[END_REF][START_REF] Ogasawara | Evaluation of Breast Lymphatic Pathways With Indocyanine Green Fluorescence Imaging in Patients With Breast Cancer[END_REF][START_REF] Stummer | Fluorescence-Guided Surgery With 5-Aminolevulinic Acid for Resection of Malignant Glioma: a Randomised Controlled Multicentre Phase III Trial[END_REF] studies have shown the potential clinical use of NIRF imaging to improve the therapeutic outcome of surgery. Compared to light in the visible spectral range (400-650 nm), application of near-infrared (NIR) light minimizes absorption by physiologically abundant molecules such as hemoglobin and lipids, which increases penetration depth (16;17). Additionally, autofluorescence (the intrinsic fluorescence signal present in all living cells due to various normal metabolites and tissue constituents) is strongly reduced in the NIR spectral range.

Taken together, these aspects of NIR light make it particularly suitable for use in

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intraoperative optical imaging applications. However, clinical application of NIRF imaging in BCS is currently limited to the non-specific intraoperative detection of the sentinel lymph node (11;12;14;18-20).

Tumour-targeted near-infrared fluorophores

With the introduction of clinical grade tumour-targeted NIR fluorophores, NIRF imaging may be extended towards the intraoperative detection of the primary tumour [START_REF] Keereweer | Optical Image-Guided Surgery-Where Do We Stand?[END_REF]. Several target molecules have been indentified for breast cancer that may be of value for such an approach, including Her2/neu receptor (9;21;22), vascular endothelial growth factor (VEGF) receptor (23;24), endothelial growth factor (EGF) receptor [START_REF] Sampath | Dual-Labeled Trastuzumab-Based Imaging Agent for the Detection of Human Epidermal Growth Factor Receptor 2 Overexpression in Breast Cancer[END_REF] and folate receptor-α [START_REF] Sega | Tumor Detection Using Folate Receptor-Targeted Imaging Agents[END_REF].

In tumour-targeted NIRF imaging, a tumour-targeted NIR fluorophore is administered several hours or days prior to the imaging procedure. Subsequently, an external laser is used to irradiate the breast with light in the NIR spectral range (650-900 nm) [START_REF] Ntziachristos | Fluorescence Molecular Imaging[END_REF].

Upon excitation, the fluorophore will release photons of a higher NIR wavelength.

Because NIR light is invisible to the naked eye, a dedicated optical imaging system is necessary to capture the NIR signal from the surgical field and digitally convert it to a visible image. Recently, we and our co-workers developed a multispectral NIRF intraoperative camera system that is suitable for intraoperative use with NIR fluorophores [START_REF] Themelis | Real-Time Intraoperative Fluorescence Imaging System Using Light-Absorption Correction[END_REF].

Simulation of NIRF-guided breast-conserving surgery

In the current preclinical study, we evaluated intraoperative NIRF imaging applications in a simulated clinical setting as a step-up toward NIRF-guided BCS. To this end, we used tissue-simulating gelatin-based breast phantoms that mimic the

M A N U S C R I P T A C C E P T E D

optical properties of normal breast tissue (28;29). Tumour-like fluorescent inclusions of different size and shape were positioned at predefined sites in the phantoms, allowing for simulation of i) preoperative tumour localization, ii) real-time NIRF-guided tumour resection and iii) intraoperative macroscopic margin assessment. The tumorlike inclusions contain the non-specific NIR fluorophore indocyanine green (ICG), to simulate for the use of tumour-targeted near-infrared fluorophores in BCS. Currently, ICG (absorption and emission maximum at ~780 and ~820 nm, respectively) is one of the few FDA-approved NIR fluorophores available for clinical use [START_REF] Chopra | Trastuzumab Complexed to Near-Infrared Fluorophore Indocyanine Green[END_REF]. Sevick-Muraca et al. have previously shown the feasibility of NIRF imaging following microdose administration of ICG [START_REF] Sevick-Muraca | Imaging of Lymph Flow in Breast Cancer Patients After Microdose Administration of a Near-Infrared Fluorophore: Feasibility Study[END_REF]. Although ICG in itself is non-specific, their findings suggest that comparable microdose concentrations can be used to label cancer cells with tumour-targeted NIR fluorophores for intraoperative NIRF imaging.

Importantly, new fluorophores in the NIR spectral range are currently being developed, e.g. IRDye ® 800CW, with properties more promising for intraoperative use compared to ICG [START_REF] Sampath | Dual-Labeled Trastuzumab-Based Imaging Agent for the Detection of Human Epidermal Growth Factor Receptor 2 Overexpression in Breast Cancer[END_REF].

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Material and Methods

Assessment of ICG fluorescence self-quenching in agarose

Because increasing concentrations of ICG may not correspond to an increased fluorescence signal due to self-quenching of ICG, different concentrations of ICG in agarose were evaluated for fluorescence activity (29;30). Briefly, an ICG stock solution was serially diluted in 10 ml sterile water (ranging from 0.5 uM to 350 uM ICG), after which 2% agarose was added. The mixture was then heated to 70°C and stirred until the agarose was completely dissolved. After solidification of the agarose mixture for 15 min at 4°C, fluorescence reflectance imaging (FRI) was performed to determine maximum photon counts/sec (settings: exposure time: 1000 ms, excitation: 780 nm, emission: 820 nm).

Assessment maximal penetration depth of ICG fluorescence

In order to determine the maximal penetration depth of the NIRF signal, a cubic fluorescent inclusion of 5x5x5 mm containing 14 µM ICG was positioned in phantom tissue at a depth of 30 mm. Subsequently, the surgeon excised 3-4 mm layers of phantom towards the inclusion (remaining depths were 27, 24, 21, 18, 15, 11, 7, and 4 mm, respectively). At all depths, FRI was performed with the intraoperative NIRF camera system (exposure time: 3000-60000 ms, excitation 780 nm, emission 820 nm, binning: small-medium). Maximum photon counts per second exposure time were calculated as well as the full width at half maximum (FWHM) of the fluorescence signal. The FWHM is a measure for scattering and indicates the diameter of the fluorescence signal when the intensity of the signal is reduced to half the maximum. Scattering both contributes to signal loss and loss in resolution. The

Tissue-Simulating Breast Phantoms

Composition of the tissue-simulating gelatin-based breast phantoms was aimed at obtaining uniform optical properties that closely match the optical characteristics of normal breast tissue, as described in detail before [START_REF] Grand | Tissue-Like Phantoms for Near-Infrared Fluorescence Imaging System Assessment and the Training of Surgeons[END_REF]. Additionally, the breast phantoms mimic the elastic properties of human tissue [START_REF] Lizuka | Optical Phantom Materials for Near Infrared Laser Photocoagulation Studies[END_REF].

Briefly, 10% gelatin 250 (Natural Spices, Watergang, the Netherlands) was dissolved in 1 l TBS (50 mmol Tris-HCl ,150 mmol NaCl , pH 7.4). To remove molecular oxygen and prevent microbial infection, 15 mmol NaN 3 (Merck, Darmstadt, Germany) was added. The gelatin slurry was completely dissolved by heating to 50°C and subsequently cooled down to 35°C and maintained at this temperature. Under constant stirring, 170 µmol hemoglobin (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 1% Intralipid ® 20% (Sigma-Aldrich) were added. Next, the gelatin mixture was poured in a custom-made pre-chilled breast-shaped mold (end volume 500 ml) to a level that corresponded to the predefined depth of the agarose inclusion.

After solidification for 30 min at 4°C, a tumour-li ke NIR fluorescent agarose inclusion was positioned on the surface and temporarily fixed with a small needle. Next, the remaining of the warmed gelatin mixture was added to fill up the remaining mold volume, allowing for adherence of both layers. The phantom was then stored in the dark to solidify for another 30 min at 4 o C, after which it was gently removed from its mold.

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In total, 4 breast phantoms were constructed with tumour-like NIR fluorescent agarose inclusions of different size and shape (figure 1A) positioned at predefined depths. Imaging of the phantoms was performed directly after production of all 4 phantoms.

Breast phantom #1 contained 2 similar-sized (Ø1.0 cm) sphere-shaped agarose inclusions at different depths (2.0 and 4.0 cm). Phantom #2 contained 2 sphereshaped inclusions at the same depth (1.5 cm), differing in size (Ø 0.5 cm and Ø 2.0 cm). Phantom #3 contained 1 sphere-shaped (Ø1.0 cm) and 1 prolate sphere-shaped (Ø1.0 cm) agarose inclusion at the same depth (1.5 cm). Finally, phantom #4 contained 2 irregular shaped agarose inclusions of similar size (Ø1.5 cm) at different depths (1.5 and 3.0 cm).

Tumour-like NIR fluorescent agarose inclusions

For tumour-like NIR fluorescent agarose inclusions, 2% agarose (Hispanagar, Burgos, Spain) was used instead of 10% gelatin. Agarose has a higher melting point which prevents the inclusions from dissolving and leaking ICG (see below) during and after the positioning procedure in the gelatin phantom. In short, a 2% (W/V) agarose slurry was heated to 70°C and stirred until the agarose was completely dissolved. Subsequently, ICG (ICG-PULSION ® ; Pulsion Medical Systems, Munich, Germany) was dissolved to a final concentration of 14 µM. Finally, in order to resemble the optical appearance of the surrounding breast phantom tissue, 170 µM hemoglobin,15 mM NaN 3 and 1% Intralipid ® 20% were added to the tumour-like fluorescent inclusions.

Tumour-like fluorescent inclusions of different size (range: 0.5 cm to 2.0 cm) and shape (prolate sphere, sphere and irregular shape, Figure 1A) were produced. The

Near-infrared fluorescence imaging system

A custom-made NIRF camera system was developed in collaboration with SurgOptix Inc (SurgOptix Inc, Redwood Shores CA, USA) for real-time intraoperative imaging.

The system implements a correction scheme that improves the accuracy of epiillumination fluorescence images for light intensity variations in tissue.

Implementation is based on the use of three cameras operating in parallel. The camera is mounted on a five degrees of freedom bracket, the sixth degree (rotation) can be performed digitally. This camera allows for simultaneous acquisition of colour videos and normalized fluorescence images in real-time, yielding a lateral resolution up to 66.58 µm and a variable field of view (FOV) of 13.5W x 11H to 115W x 95H (mm). A description in full detail is provided by Themelis et al [START_REF] Themelis | Real-Time Intraoperative Fluorescence Imaging System Using Light-Absorption Correction[END_REF]. The invisible NIRF imaging signal was digitally converted into a pseudo-colour and superposed on a colour video image of the operating field, allowing for real-time, intraoperative anatomical positioning of the fluorescence signal.

Simulation of intraoperative NIRF imaging

Breast phantoms with tumour-like NIR fluorescent agarose inclusions were used to simulate and evaluate the potential of NIRF imaging applications in BCS (Figure 1 and 3). In all phantoms, the location of the tumour-like fluorescent inclusions was assessed preoperatively with non-invasive NIRF imaging. In phantoms #1 and #2, the tumour-like fluorescent inclusions were subsequently excised using conventional

M A N U S C R I P T A C C E P T E D

ACCEPTED MANUSCRIPT 10 surgical equipment, guided solely by visual inspection, tactile information, and preoperatively obtained NIRF imaging data. The surgeon was asked to indicate when he believed a complete excision of the tumour was reached. Subsequently, the NIRF camera system was applied to assess the feasibility of NIRF-guided macroscopic margin assessment of the surgical cavity and excised tissue fragments. In case of an incomplete excision, the surgeon was asked to perform a re-excision under real-time NIRF-guidance.

In phantoms #3 and #4, the tumour-like fluorescent inclusions were localized and excised under real-time NIRF guidance. While approaching the tumour-like fluorescent inclusions, the surgeon was supported with both visible and audible information. In short, the detected fluorescent signal was depicted on a TFT-screen and was made quantitatively audible using a digitally generated sound-pitch. In this approach, an increase in sound-pitch represents an increase in fluorescence signal indicating the approximation of a tumour-like fluorescent inclusion.

M A N U S C R I P T A C C E P T E D

Results

ICG fluorescence self-quenching in agarose

To determine the optimal ICG concentration in agarose, a serial range of increasing ICG concentrations was analyzed for fluorescence characteristics. The optimal fluorescence signal was observed at a concentration of approximately 10 µM ICG (Figure 4). These results are comparable to self-quenching characteristics of ICG as previously determined in gelatin [START_REF] Grand | Tissue-Like Phantoms for Near-Infrared Fluorescence Imaging System Assessment and the Training of Surgeons[END_REF].

Maximal penetration depth of ICG fluorescence

The maximal tissue penetration depth of a detectable fluorescent ICG inclusion was reached at a depth of 21 mm (Figure 2C and supplemental video 2).

Simulation of intraoperative NIRF imaging

Preoperative NIRF-guided localization of tumour-like fluorescent agarose inclusions was performed in 4 different breast phantoms. The various tumour-like inclusions positioned at a depth of ≤ 2.0 cm were detectable with the NIRF camera system (figure 3A-I). Tumour-like inclusions positioned at depths of 3.0 and 4.0 cm could not be detected preoperatively.

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Tumour-like inclusions in phantom #1 and #2 were excised using conventional techniques for tumour localization. In phantom #1, one out of two tumour-like inclusions proved to be only partially excised, as evidenced by a remnant strong fluorescence signal in the surgical cavity detected by the NIRF camera system (figure 3C-III). In phantom #2, the excision of one out of two inclusions was found to be incomplete. In case of residual fluorescence, the surgeon could detect and excise (theranostic procedure) the remnant inclusion under real-time NIRF guidance (figure 3C-IV and supplemental video 1). In all cases, NIRF-guided re-excision resulted in a complete excision, without the need for additional excision of large breast phantom fragments (figure 3C-V).

In phantoms #3 and #4, the tumour-like inclusions were located (figure 3B) and excised (figure 3C) under real-time NIRF-guidance. Although the inclusion at 3.0 cm depth in phantom #4 could not be detected preoperatively (figure 3B-I), it was detectable using the NIRF camera system after an incision of approximately 1 cm of superficial phantom tissue (figure 3B-II). In phantom #3, no residual tumour-like inclusion material could be detected after initial NIRF-guided excision, while the excision of one out of two irregular inclusions in phantom #4 was found to be incomplete. Again, subsequent NIRF-guided re-excision resulted in a complete excision.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 13

During the surgical simulation procedure, the approximation of the surgeon towards tumour-like fluorescent agarose inclusions was guided by both visual information on a TFT screen and audible sound-pitched information (Supplemental videos 1-2). The approach resulted in a clear change in the signal strength of the fluorescence image that was accompanied with an increase of the sound pitch at ~15 mm prior to excision of the tumour-like agarose inclusion. These signals assisted the surgeon in carefully advancing the margins.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 14

Discussion

Tissue-like phantoms and tumour-like inclusions

The composition of the breast phantoms was based on data published by De Grand et al, who developed and validated phantoms to mimic the basic optical characteristics (absorption and scattering coefficients) of breast tissue [START_REF] Grand | Tissue-Like Phantoms for Near-Infrared Fluorescence Imaging System Assessment and the Training of Surgeons[END_REF]. The absorption of photons by both cellular organelles and blood was simulated by hemoglobin, which gives the phantoms a deep red colour (32;33). Additionally, Intralipid ® was added to mimic scattering properties of breast tissue [START_REF] Pogue | Review of Tissue Simulating Phantoms for Optical Spectroscopy, Imaging and Dosimetry[END_REF].

In order to resemble the clinical situation as close as possible, tumour-like fluorescent agarose inclusions were incorporated in the breast phantoms. The agarose-based inclusions simulate the firm-elastic consistency of tumour tissue and allow for surgical margin status assessment, both intraoperatively (NIRF-guided surgery) and ex vivo (NIRF-guided macroscopic margin assessment). The relatively low concentration of ICG used in this study resembles the potential application of microdose tumourtargeted fluorophores (ranging from 1 to 100 µM) in BCS.

Although the phantoms used in this study are homogeneous, and therefore do not possess the complex structures that characterize mammary tissue, they do provide a tool for assessing the value of theoretical assumptions and indicate generally important features of future clinical NIRF imaging applications in BCS. Additionally, due to limited depth resolution and a non-linear dependence of the signal detected and the depth of the fluorescence activity, NIRF imaging by epiillumination with our current camera system seems of limited value for preoperative localization of tumours. This applies in particular to situations where the tumour is located relatively deep (> 2 cm) in fat and glandular tissue of the breast. However, since the surgeon, by definition, will bring the area of interest closer to the surface during surgery, our multispectral NIRF camera system is well-suited for intraoperative imaging applications.

NIRF imaging instruments designed for preoperative imaging, e.g. the SoftScan (ART, Advanced Research Technologies, Saint-Laurent (Quebec), Canada), show penetration depths far superior (up to 15 cm) to intraoperative imaging systems [START_REF] Intes | Time-Domain Optical Mammography SoftScan: Initial Results[END_REF]. This difference in penetration depth is due to the application of different imaging strategies which are largely incompatible with surgery, e.g. trans-illumination and the need for light conduction liquid media.

Non-specific versus tumour-targeted fluorophores

Several possibilities exist for delivering fluorophores to the tumour. One possibility would be to inject a non-specific fluorophore (e.g. ICG) into the tumour under stereotactic or ultrasonographic guidance [START_REF] Berridge | Indocyanine Green Dye As a Tissue Marker for Localization of Nonpalpable Breast Lesions[END_REF]. However, there are some significant drawbacks to this approach. First, the injection of the nonspecific fluorophore into the tumour is a critical step in the procedure and has to be very accurate to minimize false-negative and false-positive results. Additionally, spillage/leakage of fluorophore within the mammary gland during the procedure will decrease accuracy of both localization of the tumour and macroscopic margin assessment. Therefore, we believe NIRF-guided surgery should ideally be combined with tumour-targeted fluorophores, which provide molecularly-specific detection of cancer cells. In these agents, the NIR fluorophore has been conjugated to a specific targeting ligand or monoclonal antibody. This allows for tumour-specific binding of the fluorophore, increasing SN ratios and minimizing spillage of the fluorophore during the surgical procedure (3;10). Several studies have shown the feasibility of using tumour-targeted fluorophores in vivo to image tumours intraoperatively, including the use of tumourtargeted ICG-conjugated agents (5;9;22;25;26;36;37). However, there are some significant drawbacks, including the heterogeneity of (breast) tumours which should be solved before applying tumour-targeted NIRF imaging in the clinic. In BCS, the preoperative biopsy taken prior to surgery could provide important information on molecular targets for NIRF imaging. As this biopsy is considered standard practice, it will not require an additional invasive procedure, while offering the possibility to determine the expression of different kinds of molecular targets present on the breast cancer cells by immunohistochemical analysis. The surgeon could then look for NIRF agents suited for each individual tumour, offering a more patient-tailored approach.

Conclusion

We have pre-clinically assessed the applicability of NIRF imaging applications in BCS by exploiting tissue-simulating breast phantoms. NIRF-guided intraoperative tumour localization and detection of remnant disease showed feasible. Clinical studies are needed to further validate these results for use in BCS.

7 FWHM

 7 indicates the minimal distance between two distinct sources to be recognized as separate.

  in the breast phantoms as indicated and chilled in the dark for 30 min at 4°C. Imaging of each individual breas t phantom was performed within 6 h.

  Near-infrared fluorescence imaging: strengths and drawbacks NIRF imaging offers a promising technique for real-time NIRF-guided surgery in BCS with little interference in the standard surgical procedure or changes in the design of the operating theatre. The technology is considered safe, fast, makes use of non-M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 15 ionizing radiation, and has a high resolution (3;10). However, NIRF imaging does have limitations originating from the intrinsic characteristics of light propagation through tissue, including scattering and absorbance (17).

Acknowledgements

This work was supported by a grant from the Jan Kornelis de Cock foundation. The authors wish to thank Mr. T. Buddingh, MD for assistance in operating on the phantoms.


T M Gulik Van

J J Kloek

A T Ruys

O R C Busch

G J Van Tienhoven

J S Lameris

E A J Rauws

D J Gouma

T M Gulik

Gulik Van Tm

Kloek Jj

Ruys At

Busch Orc

Tienhoven Gj

MD Thomas M Van Gulik
email: t.m.vangulik@amc.uva.nl

Multidisciplinary management of hilar cholangiocarcinoma (Klatskin tumor): extended resection is associated with improved

published or not. The documents may come     Multidisciplinary management of hilar cholangiocarcinoma (Klatskin tumor): extended resection is associated with improved survival

Introduction

The improved surgical treatment of hilar cholangiocarcinoma (HCCA) in the past two decades [START_REF] Ito | Hilar cholangiocarcinoma: current management[END_REF] , not only owes to changed surgical strategy, but also to better patient selection and improved preoperative work-up of candidates for resection. The synergy of endoscopists, interventional radiologists, oncologists and surgeons, has equally contributed to these improved results. [START_REF] Seyama | Long-term outcome of extended hemihepatectomy for hilar bile duct cancer with no mortality and high survival rate[END_REF] Herein we summarize the multidisciplinary experience in diagnosis and management of HCCA over a period of two decades at the Academic Medical Center in Amsterdam, with emphasis on surgical outcome.

Patients and methods

Study population

From 1988 until 2003, 117 consecutive patients underwent resection on the suspicion of HCCA. These patients were considered according to three 5-year time periods, i.e. period 1 (1988-1993), 2 (1993-1998) and 3 (1998-2003). A full 5-year follow-up was obtained for the last group which has currently been analysed in patients with microscopically proven HCCA.

Outcomes were evaluated by assessment of completeness of resection, postoperative morbidity and mortality.

Diagnosis and staging

Suspicion on HCCA was usually based on clinical presentation and imaging studies.

Diagnosis could be confirmed by (brush) cytology but, microscopical evidence was not prerequisite for undergoing resection.

The Bismuth-Corlette classification was used to stage proximal tumor infiltration into the biliary tract. [START_REF] Ito | Hilar cholangiocarcinoma: current management[END_REF] Although direct cholangiography was the gold standard diagnostic procedure in the staging of HCCA, non-invasive diagnostic investigations as MRCP (magnetic resonance cholangiopancreatography) were preferred in more recent years. Cross-sectional imaging studies such as CT and MRI were used to assess liver parenchymal invasion, vascular invasion in portal vein and/or hepatic arteries and hepatic or extrahepatic metastases.

Ultrasound, in combination with Duplex evaluation of the portal venous and hepatic arterial systems also proved useful. [START_REF] Van Gulik | Changing perspectives in the assessment of resectability of hilar cholangiocarcinoma[END_REF]

Criteria for resectability

Resectability of HCCA was in the first place determined by proximal extent of tumor into the intrahepatic biliary tree. Whereas Bismuth type III tumors showing extension into the first segmental biliary ducts on one side of the liver are resectable using (extended) hemihepatectomy, Bismuth type IV tumors which involve the segmental ducts on both sides of the liver, are resectable in selected cases.

Extra-hepatic metastases were usually a contraindication for resection. In the first two periods, the intraoperative finding of lymphnode metastases was a reason to abandon resection. In the last period, resection was carried out when lymphnode metastases were confined to the hepatic pedicle or the hepatoduodenal ligament. Tumor positive lymphnodes along the common hepatic artery or celiac axis were always considered a contraindication for resection. The portal vein bifurcation was resected along with the tumor when invasion was present. Massive involvement of the main trunk of the portal vein and/or hepatic artery precluded resection. Volume (>40%) and function of future remnant liver and the possibility of portal vein embolization were also considered.

Preoperative work-up

Staging laparoscopy was performed in the assessment of resectability, as we had shown that a laparotomy can be avoided in 25-40% of our patients with HCCA, mostly because of the detection of small peritoneal or liver metastases. [START_REF] Tilleman | Diagnostic laparoscopy and laparoscopic ultrasound for staging of patients with malignant proximal bile duct obstruction[END_REF] Similar results of staging laparoscopy in HCCA have been reported by other centers. [START_REF] Joseph | Staging laparoscopy for cholangiocarcinoma[END_REF] Preoperative biliary drainage of at least the future liver remnant was performed to ensure optimal postoperative function and regeneration of the liver remnant.

CT volumetry was used to assess the volume of the remnant liver relative to total liver volume, mainly in the last period. When the volume of the future remnant liver was less than 40%, preoperative portal vein embolization (PVE) was considered.

To prevent postoperative implantation metastases, low-dose irradiation (3.5Gy on 3 consecutive days until operation) was applied in patients with potentially resectable hilar tumors as of 1990.

The algorithm shown in figure 1 summarizes diagnosis and treatment of patients with HCCA in our center.

Type of resection

Local resection was the technique mainly used in the first two periods (1988-1998). The bile ducts at the liver hilum were dissected and transected proximally at the level of the segmental biliary ducts, and distally in the CBD.

A more extensive surgical approach as proposed by Japanese surgeons, has been applied in our center as of 1998, in which hilar resection was combined with large liver resections for the majority of HCCA. [START_REF] Van Gulik | Original article: new surgical approaches to the Klatskin tumour[END_REF] According to this strategy, radical resection of HCCA encompassed excision of the liver hilum en bloc with (extended) hemihepatectomy including the caudate lobe, excision of the portal vein bifurcation when involved and complete lymphadenectomy of the hepatoduodenal ligament. [START_REF] Dinant | The importance of complete excision of the caudate lobe in resection of hilar cholangiocarcinoma[END_REF] For biliary reconstruction, end-to-side anastmoses of the segmental ducts and a Roux-en-Y jejeunal loop were constructed.

Survival and statistical analysis

Actuarial 5-year survival data were analysed by constructing survival curves using the Kaplan-Meier method. The log-rank significance test was used for comparison of survival between groups. Factors influencing survival were analysed using univariate analysis. SPSS 16.0 for Windows (SPSS Inc, Chicago, Ill) was used as statistical software and a p value < 0.05 was considered significant.

Results

Outcomes of resection

Of all 117 patients who had undergone resection, 18 patients (15.3%) appeared to have a benign lesion on microscopical evaluation of the specimen, leaving 99 patients with histologically proven HCCA. The latter patients were divided according to the period of resection: period 1 (1988-1993) n=45; period 2 (1993-1998) n=25 and period 3 (1998-2003) n=29.

More hilar resections were combined with partial hepatectomies, especially during the third time period. (Table 1) Complete resection of segment I was performed in 15 patients 7 , all in the third period, and portal vein reconstruction was performed in 7 patients, of which 6 patients in the last period [START_REF] Dinant | Improved outcome of resection of hilar cholangiocarcinoma (Klatskin tumor)[END_REF] . With this more extensive surgical approach, the proportion of margin negative resections increased from 13% in period 1 to 59% in period 3 (p<0.05).

Table 2 shows the stages of tumors resected in each period, according to the American Joint Committee (AJCC) on Cancer and Union Internationale Contre le Cancer (UICC).

Survival

Actuarial five-year survival increased significantly from 20±5% for the periods 1 and 2, to 33±9% in period 3 (P<0.05). (Fig. 2) Postoperative morbidity and mortality were considerable but did not increase with more extensive surgical strategy (68% and 10%, respectively, in period 3). The period of resection was associated with survival in univariate analysis. Hence, mainly in the last 5-year period when a more aggressive surgical approach was carried out, more hilar resections were combined with partial liver resections including segments 1 and 4, leading to a higher rate of R0 resections and improved survival.

Discussion

Difficulty in diagnosis

HCCA is a difficult tumor in terms of diagnosis and management. The diagnosis may be confirmed using brush or needle cytology. Sensitivity is however, limited due to failure to obtain a representative microscopical specimen. For these reasons, microscopical evidence of the tumor is often not available. Many of our patients are, therefore, operated without confirmed diagnosis and currently, up to 15% of resected tumors are ultimately diagnosed as benign, usually inflammatory lesions. [START_REF] Erdogan | Immunoglobulin G4-related sclerosing cholangitis in patients resected for presumed malignant bile duct strictures[END_REF] Similar experiences have been published, reporting incidences of benign lesions in up to 18% of cases resected on the suspicion of HCCA. [START_REF] Kloek | Differentiation of malignant and benign proximal bile duct strictures: The diagnostic dilemma[END_REF] Despite more advanced imaging techniques, the incidence of benign biliary strictures mimicking malignant strictures at the liver hilum has not decreased in the past ten years in our center. We recently detected lymphoplasmacellular infiltrates in lesions of 47% of the patients who were misdiagnosed as HCCA, which is compatible with an autoimmune type of cholangitis. This inflammatory condition has an analogy with IgG4-related, lymphoplasmacytic sclerosing pancreatitis as has been described in pancreatic ductal strictures. [START_REF] Erdogan | Immunoglobulin G4-related sclerosing cholangitis in patients resected for presumed malignant bile duct strictures[END_REF] Since tests that can reliably distinguish between benign and malignant hilar strictures are lacking, and complete resection is the only curative treatment for HCCA, every suspicious lesion at the hepatic hilum should be considered malignant unless proven otherwise. We accept with this policy, that 10-15% of patients with benign strictures are exposed to major surgery. On the other hand, long-term relief of bile duct obstruction is offered in this category of patients who usually are difficult to manage endoscopically or by percutaneous biliary drainage. Several techniques have emerged to better discriminate benign from malignant hilar lesions. Peroral or percutaneous cholangioscopic biopsy, endoscopic ultrasound and intraductal ultrasound in combination with biliary brushing have shown promising results, however require specific expertise. [START_REF] Nimura | Staging cholangiocarcinoma by cholangioscopy[END_REF]

Resectability of tumor determined by biliary anatomy at the liver hilum

The anatomy of the biliary ducts at the hepatic duct confluence ultimately determines whether tumor free ductal margins can be obtained with preservation of sufficient remnant liver in Bismuth type IV tumors. [START_REF] Van Gulik | Original article: new surgical approaches to the Klatskin tumour[END_REF] The liver hilum features great variation in the anatomy of the biliary ducts. The hepatic duct confluence consists in 16% of cases of the right anterior (B5/8) and right posterior (B6/7) sectorial ducts draining together with the left hepatic duct into the common hepatic duct, forming a trifurcation. Invasion of the left segmental ducts in combination with the right sectorial ducts at the liver hilum, although by definition a Bismuth type IV tumor, does not preclude a potentially curative resection. It is therefore crucial that each patient is assessed on an individual basis by an experienced multidisciplinary team.

Preoperative biliary drainage

Preoperative biliary drainage in HCCA is controversial. Obstructive jaundice affects liver functional reserve and reduces the regenerative capacity of the liver after resection. Jaundice for these reasons, is considered a significant risk factor in patients with HCCA requiring major liver resection. Although we recently showed in a randomized, multicentric trial, that preoperative biliary drainage had no beneficial effects in patients with distal bile duct carcinoma undergoing subtotal pancreatoduodenectomy, the discussion is different in patients with HCCA requiring extensive liver resection. [START_REF] Van Der Gaag | Preoperative biliary drainage for cancer of the head of the pancreas[END_REF] The advantages of preoperative biliary drainage in the setting of HCCA are the treatment of segmental cholangitis, definition of proximal extent of the tumor and improved function and regenerative capacity of the remnant liver. [START_REF] Nimura | Preoperative biliary drainage before resection for cholangiocarcinoma (Pro)[END_REF] Drawbacks are the risk of thrombosis, hemobilia, infectious complications and tumor cell seeding. [START_REF] Laurent | Cholangiocarcinoma: preoperative biliary drainage (Con)[END_REF] Because the advantages of preoperative biliary drainage outweigh the disadvantages, we pursue preoperative biliary drainage of at least the future liver remnant in patients with potentially resectable HCCA. In our experience, percutaneous transhepatic biliary drainage outperformed endoscopic biliary drainage, showing fewer infectious complications and less drainage procedures. [START_REF] Kloek | Endoscopic and percutaneous preoperative biliary drainage in patients with suspected hilar cholangiocarcinoma[END_REF]

Volume assessment of future liver remnant

Although in patients with normal liver parenchyma, 25-30% of remnant liver suffices to ensure sufficient postoperative liver function, livers in jaundiced patients such as in HCCA, are seriously compromised, even after recent biliary drainage. Therefore, in patients with HCCA, we regard 40% volume as the safe lower limit in a decompressed liver. Calculation of volume based on CT however, only provides indirect information on the functional capacity of the liver remnant. We therefore recently use 99m Tc-mebrofenin scintigraphy in conjunction with CT volumetry to determine function of the future remnant liver. This quantitative liver function test was shown to correlate with clinical outcome after resection, however, needs additional validation in other centers. [START_REF] De Graaf | Assessment of future remnant liver function using hepatobiliary scintigraphy in patients undergoing major liver resection[END_REF] When future remnant liver volume or function is deemed insufficient, preoperative portal vein embolization (PVE) is considered. [START_REF] Nagino | Two Hundred Forty Consecutive Portal Vein Embolizations Before Extended Hepatectomy for Biliary Cancer: Surgical Outcome and Long-term Follow-Up[END_REF] In our experience however, there are certain drawbacks of the use of PVE in patients with HCCA. PVE predetermines the side of the liver to be resected, and this cannot be changed when during exploration the type of resection is reconsidered on the basis of intraoperative findings.

In addition, when the patient is found to be unresectable, the persisting embolized liver segments may give rise to septic complications since the affected bile ducts are often infected and incompletely drained. [START_REF] Van Gulik | Controversies in the use of portal vein embolization[END_REF]

Preoperative radiation therapy to prevent seeding metastases

The bile of patients with HCCA is contaminated with tumor cells continuously exfoliating from the epithelial tumor. These tumor cells may give rise to seeding metastases as is a known complication of percutaneous biliary drainage in which seeding metastases occur along the track of the tube, as reported in up to 6% of cases. [START_REF] Laurent | Cholangiocarcinoma: preoperative biliary drainage (Con)[END_REF] The risk of free tumor cells in the bile is enhanced after pertubation while during resection, bile spill potentially leads to seeding metastases in the operative field. Endoscopic stenting for drainage in patients with HCCA in our center, was associated with a significantly higher risk of implantation metastases presenting in the drain track scar or laparotomy scar following resection. [START_REF] Ten Hoopen-Neumann | Occurrence of implantation metastases after resection of Klatskin tumors[END_REF] Therefore, to destruct tumor cells contained in the bile, we preoperatively apply low-dose irradiation (3x3.5Gy) in patients with potentially resectable hilar tumors. Since this protocol was used, implantation metastases were not anymore encountered in 82 consecutive patients followedup after resection after previous (endoscopic) biliary drainage. [START_REF] Ten Hoopen-Neumann | Occurrence of implantation metastases after resection of Klatskin tumors[END_REF] Of note is that more recent surgical refinements have controlled bile spill during resection and may as well have contributed to the decreased risk of postoperative seeding metastases. After transection of the common bile duct, the stump is meticulously closed to avoid bile spill whereas the proximal, segmental bile ducts are cut in the liver remnant after the parenchymal transection has been completed.

The extent of resection

Most HCCA (type II, III, and IV) require hilar resection in combination with substantial resection of liver parenchyma. The single most important prognostic factor for long-term survival is complete (R0) resection of the tumor. [START_REF] Kloek | Surgery for extrahepatic cholangiocarcinoma: predictors of survival[END_REF] Margin negative resections are however difficult to achieve due to the central location of the tumor at the liver hilum and proximal infiltration into the segmental bile ducts. The right and left portal vein and the hepatic artery branches run in direct proximity of the tumor. Surgery with curative intent therefore requires hilar resection in combination with extended liver resection, often in combination with excision of the unilateral portal vein and bifurcation. Several series have shown that these extensive resections lead to an increased rate of R0 resections and hence, improved survival. 1;2;21-23 The concept of hilar resection in conjunction with liver resection is based on a threedimensional perception of the tumor located centrally in the liver. Tumor extension occurs from the bile duct confluence to the right and left along the main hepatic and segmental bile ducts. In addition, the tumor potentially invades anteriorly into the duct(s) of segment 4 and posteriorly, into the bile ducts draining segment 1. It is therefore crucial that the central sector of the liver along the antero-posterior axis including segments 1 and 4 is considered with resection. A right-sided resection therefore, usually entails an extended right hemihepatectomy including the segments 4 and 1, leaving only segments 2 and 3 as liver remnant. There is an advantage of a left-sided approach of resection since segment 4 is an anatomical part of the left liver, hence preserving most of the right liver.

The Berlin surgical group advocates a no-touch technique following oncological rules to resect right-sided HCCA in conjunction with extended liver resection. The surgical strategy consists of performing hilar resection en bloc with extended right hemihepatectomy and unconditional resection of the portal vein bifurcation followed by end-to-end reconstruction.

Dissection of the portal vein in the direct vicinity of the tumor is hence avoided. In a selection of patients operated according to these oncological principles, the 5-year survival rate was 57%. [START_REF] Neuhaus | Radical surgery for right-sided klatskin tumor[END_REF]

Postoperative morbidity and mortality

Hilar resection in combination with extensive liver resections are undertaken at the cost of considerable morbidity and mortality (68% and 10%, respectively, in period 3 of this series).

Most series report a hospital mortality of 5-10%. The challenge for the near future is to decrease mortality of the extended liver resections that are necessary to radically remove the tumor. The most important cause of postoperative mortality is liver failure. [START_REF] Neuhaus | Radical surgery for right-sided klatskin tumor[END_REF] Hence, quantitative assessment of volume and function of the future remnant liver is crucial in the evaluation of patients undergoing large liver resections for HCCA.

Survival

Overall, 5-year survival rates of 20% up to 35% have been reported after resection of HCCA. 1;2;21;22;25 The nearly 34% 5-year survival rate of our patients in the last group compares favorably with these results. The most important independent prognostic factor for long-term survival was the period of resection. The Mayo clinic has reported good results of the combination of chemoradiation and orthotopic liver transplantation (OLT) in patients with HCCA presenting with unresectable disease. [START_REF] Rosen | Surgery for cholangiocarcinoma: the role of liver transplantation[END_REF] In a series of highly selected patients, 5-year actuarial survival for all patients that began neoadjuvant therapy was 55%, and 5-year survival after OLT was 71%. This experience needs to be confirmed in additional liver transplant centers but readdresses the potential of total hepatectomy and liver transplantation in the treatment of HCCA. [START_REF] Rosen | Surgery for cholangiocarcinoma: the role of liver transplantation[END_REF]

Adjuvant treatment

The role of additional therapy after resection in patients with hilar cholangiocarcinoma is controversial. The success of neoadjuvant chemoradiation in patients with advanced HCCA according to the Mayo clinic experience has renewed interest in the use of chemoradiation in patients with resectable HCCA. So far, however, no adjuvant regimens have shown a survival benefit. Postoperative radiotherapy was part of our treatment protocol as of 1983. We previously analysed 91 patients who had undergone resection between 1983 and 1998, of which 20 patients had no additional radiotherapy, 30 patients had only external radiotherapy (46 ± 11 Gy) and 41 patients had a combination of external (42 ± 5 Gy) and intraluminal brachytherapy (10 ± 2 Gy). [START_REF] Gerhards | Results of postoperative radiotherapy for resectable hilar cholangiocarcinoma[END_REF] Median survival after treatment with adjuvant radiotherapy was significantly longer than after resection without additional radiation (24 months vs. 8 months, respectively). To investigate whether the retrospectively observed survival benefit was real, a randomized trial was attempted but this failed due to insufficient accrual. Because of the lack of evidence and the additional toxicity of postoperative irradiation, the trial was abandoned in 2004. Recent experimental studies have shown encouraging results of new chemotherapy regimens including targeted therapy with anti-EGFR and antiangiogenic drugs. [START_REF] Hezel | Systemic therapy for biliary tract cancers[END_REF] Photodynamic therapy (PDT) holds promise as a novel treatment in patients with advanced disease. A randomized study in patients with unresectable type III and type IV tumors showed that PDT improved survival, decreased cholestasis and improved quality of life scores as compared to stenting alone. In the neoadjuvant setting, patients with advanced HCCA underwent preoperative PDT followed by a potentially curative (R0) resection. [START_REF] Zoepf | Photodynamic therapy of cholangiocarcinoma[END_REF] The benefits of PDT await further confirmation in randomized studies.

Conclusion

The multidisciplinary efforts of all specialities involved in the treatment of HCCA, has culminated in an institutional expertise that has greatly improved the surgical results of patients with HCCA. A more aggressive surgical approach applied in our center as of 1998, has contributed to an increased rate of R0 resections and significantly improved survival. The outcomes support the plea that patients with this rare and complex tumor are managed in highly specialized centers.

Legends Figure 1

Algorithm for multidisciplinary management of patients with hilar cholangiocarcinoma (HCCA) Actuarial five-year survival increased significantly from 20±5% to 33±9%, for the first and last period, respectively (log-rank test, p<0.05).

Table 1. Types of resection and R0 resection rate in 99 patients with hilar cholangiocarcinoma according to resection period. More hilar resections were combined with partial hepatectomies, segment 1 resections and portal vein reconstructions in the last time period, leading to a higher rate of R0 resections..

1988-

Figure 2 Kaplan-

 2 Figure 2

Table 2 .

 2 Staging according to AJCC/UICC, of 99 patients with hilar cholangiocarcinoma divided into resection period.

	1993	1993-1998	1998-2003	Total
	n=45	n=25	n=29	n=99