Dying at home or in an institution : perspectives of Dutch physicians and bereaved relatives Introduction Previous studies have shown that most people prefer to die at their own home . We investigated whether physicians or bereaved relatives in retrospect differently appreciate the dying of patients in an institution or at home . Introduction The two most important events in life , that is , birth and death , relatively often occur at home in The Netherlands . Over the period 1995 -- 2000 , around one third of all Dutch births were home deliveries -LSB- 1 -RSB- . Further , in 2001 , about 40 % of all deaths have been found to occur at home , 40 % in hospital , and about 20 % in nursing homes -LSB- 2 -RSB- . Medical care in The Netherlands is strongly founded on home-based general practice . General practitioners or family physicians provide all basic medical care to outpatients and serve as a gate door to specialized care for patients with more complex health problems . In 2003 , around 45 % of deaths due to cancer occurred at home in The Netherlands -LSB- 3 -RSB- . In the UK , the percentage of cancer deaths at home is lower and falling , from 27 % in 1994 to 22 % in 2003 -LSB- 4 -RSB- . In a study of the place of death of cancer patients in the Houston area , USA , 35 % died at home -LSB- 5 -RSB- . End-of-life care is thus , especially in The Netherlands , rather often provided in the home situation by general practitioners , home-care nurses , and informal caregivers . Research has shown that dying at home is usually preferred over dying in an institution , in The Netherlands as well as in other countries -LSB- 6 -RSB- . Such a preference seems to be predominantly shaped by whether or not people have sufficient informal carer resources -LSB- 7 -RSB- . In contrast , people who are concerned about the medical management of their symptoms may appreciate the easy access to professional caregivers in an institutional setting . Concerns about burdening relatives have also been found to contribute to a wish to die in an institution -LSB- 7 -RSB- . The finding that people 's wish to die at home becomes less predominant when death is nearing may be related to such concerns , as heavily burdening relatives with care duties is one of the most commonly recognized threats to a peaceful dying process -LSB- 6 -RSB- . A gradual shift in preference may also be due to the growing awareness of seriously ill people that dying sometimes involves problems and symptoms that are best treated by professional caregivers in an institutional setting . Finally , differential experiences of services also influence people 's preferences -LSB- 7 -RSB- . Enabling people to make genuine choices about their end-of-life care and to die at the place they prefer is often seen as a major challenge to current end-of-life care -LSB- 8 -- 10 -RSB- . Home-care patients have been reported to have more control over the effects of their illness , medical care , and treatment received than patients receiving institutional care -LSB- 11 -RSB- . However , it is unclear if the relatively high home death rate in The Netherlands is really beneficial to the quality of death and dying . Little is known about the experiences of dying patients and their caregivers in different health care settings . We investigated to what extent dying in an institution or at home involves differences in care and its appreciation by physicians and bereaved relatives . Materials and methods Patients This study concerns a sub-sample of a cohort of 128 patients with advanced breast cancer , colorectal cancer , ovary cancer , lung cancer , or prostate cancer , who were followed for a study on end-of-life care needs and practices during the last stage of their lives -LSB- 12 -RSB- . Of these patients , 103 died within the time frame during which we were able to approach attending clinical specialists , general practitioners , and bereaved relatives for an after-death data collection . In 102 cases , physicians were willing to fill out a written questionnaire on the medical treatment and dying process of these patients : Questionnaires were filled out by clinical specialists in 30 cases , by general practitioners in 17 cases , and by both in 55 cases . Relatives who had been closely involved with the patient could be contacted in 91 cases ; 63 gave their written consent to be personally interviewed at their own home . The reason for not participating was most often that relatives expected participation to be too burdensome . The patients for whom bereaved relatives participated in an interview had on average a longer disease duration -LRB- 33 vs 21 months ; p = 0.03 -RRB- and were more often women -LRB- 59 vs 28 % ; p = 0.004 -RRB- than other patients . The Medical Ethical Committee of the Erasmus MC , University Medical Center Rotterdam approved the research protocol . Questionnaire for attending physicians As soon as we were informed of the death of a patient , physicians received a self-developed written questionnaire addressing the end-of-life medical treatment and decision-making . The median time between the patient 's death and filling out the questionnaire was 12.5 weeks -LRB- range , 1 to 59 weeks -RRB- . The questions on end-of-life decision-making were based upon questionnaires that have proven to yield valid information in previous studies -LSB- 2 , 13 -RSB- . In cases where both a clinical specialist and a general practitioner filled out a questionnaire , the information about the patients ' anti-tumor treatment history , medical decision-making , and any ` negative ' aspects of the dying process were considered to be additive . Interview with bereaved relatives Interviewers were trained to carry out interviews with vulnerable people during a 2-day course . The interviews were on average held 4.9 months after the patient 's death -LRB- range , 0.8 -- 9.5 months -RRB- . The interview schedule included the following topics : personal characteristics of the bereaved relatives , such as age , sex , and relationship with the patient ; symptoms of the patient , that is , loss of appetite , pain , fatigue , dyspnoea , nausea , mouth or mucous problems , incontinence , bedsores , confusion , anxiety , and depression ; and whether or not the patient was treated for these symptoms . The patient symptom list was based upon the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 -LSB- 14 , 15 -RSB- and the Problems and Needs in Palliative Care questionnaire -LSB- 16 , 17 -RSB- and completed with psychological symptoms . Further , contacts with health care professionals , admission to hospital or other care institutions , the actual and preferred place of death , and problems in end-of-life care during the last 3 days of life were assessed using scales from the Voices of Informal Carers-Evaluation of Services -LRB- VOICES -RRB- questionnaire -LSB- 18 -RSB- . Finally , we asked the bereaved relatives about the burden of care giving . We adapted several questions from the Caregiver Reaction Assessment to make them applicable for an after-death interview with bereaved relatives -LSB- 19 -RSB- . Statistical analyses We compared the characteristics of patients who died in an institution with the characteristics of patients who died at home . Student 's t tests and χ2 tests were used to assess the statistical significance of differences between both groups . All analyses were done using the Statistical Package for the Social Sciences version 11.0 . Results The characteristics of all 103 deceased patients and of 63 patients for whom an after-death interview with a bereaved relative was available are presented in Table 1 . The mean age at death of all patients was 65 years ; 48 patients -LRB- 47 % -RRB- were women . The most common types of cancer were lung cancer -LRB- 48 patients -RRB- and breast cancer -LRB- 19 patients -RRB- . Of all 103 patients , 49 died within an institution : 35 patients died at a hospital department , 6 patients died after having been admitted to a department within a hospital , nursing home , or home for the elderly that was specialized in care for dying patients , 4 patients died within a general department of a nursing home or home for the elderly , and 4 patients died in a hospice . The remaining 54 patients died at home or in a home-like situation : 52 patients died in their own homes , 1 patient died in the home of a son , and 1 patient died during a holiday on a boat . The only significant difference between patients dying in an institution and patients dying at home concerned the percentage who had lived with a partner , which was 63 % for patients who died in an institution and 83 % for patients who died at home . All other characteristics of patients and relatives that are listed in Table 1 were similar in both groups . The characteristics of the group of patients for whom a relative could be interviewed were also similar to the characteristics of the total group . The interviewed relative was the patient 's spouse in 39 out of the 63 cases ; in 19 cases , it was a son or daughter , and in 5 cases , another relative . The majority of relatives were women , and their mean age was 55 years . Table 1Characteristics of patients and bereaved relativesCharacteristicsAll deceased patients -LRB- N = 103 -RRB- Deceased patients for whom a relative was interviewed -LRB- N = 63 -RRB- Patients Age at death , in years -LSB- mean -LRB- SD -RRB- -RSB- 65 -LRB- 11 -RRB- 64 -LRB- 11 -RRB- Sex -LSB- n/N -LRB- % -RRB- -RSB- Female48/103 -LRB- 47 -RRB- 37/63 -LRB- 59 -RRB- Living arrangement -LSB- n/N -LRB- % -RRB- -RSB- With partner76/103 -LRB- 74 -RRB- 47/63 -LRB- 75 -RRB- Education -LSB- n/N -LRB- % -RRB- -RSB- Lowa71/102 -LRB- 70 -RRB- 41/63 -LRB- 65 -RRB- Religion -LSB- n/N -LRB- % -RRB- -RSB- Religious62/103 -LRB- 60 -RRB- 40/63 -LRB- 63 -RRB- Urbanization -LSB- n/N -LRB- % -RRB- -RSB- Living in urban areab72/103 -LRB- 70 -RRB- 46/63 -LRB- 73 -RRB- Primary tumor site -LSB- n/N -LRB- % -RRB- -RSB- Lung48/103 -LRB- 47 -RRB- 31/63 -LRB- 49 -RRB- Breast19/103 -LRB- 18 -RRB- 14/63 -LRB- 22 -RRB- Other36/103 -LRB- 35 -RRB- 18/63 -LRB- 29 -RRB- History of anti-tumor treatment -LSB- n/N -LRB- % -RRB- -RSB- Surgery45/102 -LRB- 44 -RRB- 26/62 -LRB- 42 -RRB- Chemotherapy/hormone therapy84/101 -LRB- 83 -RRB- 51/61 -LRB- 84 -RRB- Radiotherapy54/101 -LRB- 54 -RRB- 36/61 -LRB- 59 -RRB- Place of death Institution -LSB- n/N -LRB- % -RRB- -RSB- 49/103 -LRB- 48 -RRB- 29/63 -LRB- 46 -RRB- Hospital35/4924/29 End-of-life care unit6/492/29 Hospice4/493/29 Nursing home/home for the elderly4/49 -- At home or in a home-like situation -LSB- n/N -LRB- % -RRB- -RSB- 54/103 -LRB- 52 -RRB- 34/63 -LRB- 54 -RRB- At patient 's own home52/5432/34 Elsewhere2/542/34Bereaved relatives Age at the time of dying of the patient , in years -LSB- mean -LRB- SD -RRB- -RSB- -- 55 -LRB- 15 -RRB- Sex -LSB- n/N -LRB- % -RRB- -RSB- Female -- 40/63 -LRB- 63 -RRB- Relative was patient 's -LSB- n/N -LRB- % -RRB- -RSB- : Spouse -- 39/63 -LRB- 62 -RRB- Son or daughter -- 19/63 -LRB- 30 -RRB- Other relationship -- 5/63 -LRB- 8 -RRB- aLow education : lower vocational , lower secondary general education , or primary schoolbLiving in an urban area : patients who were treated as an outpatient in a hospital inside the Rotterdam area Some aspects of medical care are shown in Table 2 . In both groups , physicians reported that about two thirds of the patients had died peacefully . Patients who died in an institution had less often -LRB- 37 % -RRB- been ready to die than patients who died at home -LRB- 71 % -RRB- . Agitation was a common problem during the dying process . Most patients in both settings had been unconscious before death . Physicians had discussed a number of end-of-life decisions each with about one third of their patients : This holds for decisions to forgo potentially life-prolonging treatment , intensive treatment of pain , and active euthanasia . Sedation was the only treatment option that was discussed slightly more often with patients who died in an institution . Life had actually been shortened due to the forgoing of potentially life-prolonging treatment or to the use of potentially life-shortening drugs in about one third of all cases in both settings . Such medical decisions were usually made with clear consent of the patient , and life was generally shortened by less than 1 week . Table 2Dying in an institution or at home : the physician 's perspective Place of deathP value χ2 testIn institution -LRB- N = 49 -RRB- At home -LRB- N = 54 -RRB- N -LRB- % -RRB- Physician 's evaluation of dying processa Patient died peacefully28/41 -LRB- 68 -RRB- 29/42 -LRB- 69 -RRB- 0.94 Patient was able to say goodbye to relatives26/40 -LRB- 65 -RRB- 33/44 -LRB- 75 -RRB- 0.32 Patient was ready to die15/41 -LRB- 37 -RRB- 31/44 -LRB- 71 -RRB- 0.002 Patient died suddenly and unexpectedly13/42 -LRB- 31 -RRB- 14/44 -LRB- 32 -RRB- 0.93 Patient 's dying was preceded by period of agitation25/40 -LRB- 63 -RRB- 29/41 -LRB- 71 -RRB- 0.43 Patient 's dying was preceded by period of unconsciousness30/39 -LRB- 77 -RRB- 26/42 -LRB- 62 -RRB- 0.14 Physician discussed with patient medical decisions that could shorten life Forgoing treatment13/49 -LRB- 27 -RRB- 15/50 -LRB- 30 -RRB- 0.70 Intensified pain treatment16/49 -LRB- 33 -RRB- 16/50 -LRB- 32 -RRB- 0.95 Sedation15/49 -LRB- 31 -RRB- 8/50 -LRB- 16 -RRB- 0.09 Euthanasia18/49 -LRB- 37 -RRB- 19/50 -LRB- 38 -RRB- 0.90 Life was possibly shortened due to Forgoing treatment15/47 -LRB- 32 -RRB- 14/52 -LRB- 27 -RRB- 0.59 With clear consent of the patient11/1212/13 Life was shortened by more than 1 week4/110/9 Use of potentially life-shortening drugs16/46 -LRB- 35 -RRB- 13/45 -LRB- 29 -RRB- 0.55 With clear consent of the patient12/149/10 With the explicit goal of shortening life1/164/13 Life was shortened by more than 1 week1/132/10Information as provided by clinical specialist -LRB- n = 85 -RRB- and/or general practitioner -LRB- n = 72 -RRB- . In case of conflicting answers concerning history of treatment , the information as provided by the clinical specialist prevailed . In all other cases , both sources were considered valid.aStatement was considered true if neither the clinical specialist nor the general practitioner considered it untrue . Bereaved relatives reported in 25 out of 63 cases that the patient had preferred to die at home -LRB- Table 3 -RRB- . Two patients had preferred to die in the hospital , 3 in a hospice , 2 elsewhere , and for 31 patients , the relative indicated that the patient had no clear preference . Most patients who had a preference died at the place they preferred , except for five patients who preferred home but died in the hospital . Patients who died in an institution had stayed there for on average 7 days . The number of transfers during 3 months before death was larger for patients who died in an institution -LRB- mean 1.9 -RRB- than for patients who died at home -LRB- mean 1.2 -RRB- , and the number of medical specialties that were involved with the patient was also larger in institutions -LRB- mean 2.2 -RRB- than at home -LRB- mean 1.8 -RRB- . Care involved on average six disciplines -LRB- medical and non-medical -RRB- , both in institutions and at home . Table 3Preferred and actual place of deathPreferred place of deathaActual place of deathTotal -LRB- N = 63 -RRB- In institutionAt homeHospital -LRB- N = 24 -RRB- Hospice/end-of-life care unit -LRB- N = 5 -RRB- At patient 's own home -LRB- N = 32 -RRB- Elsewhere -LRB- N = 2 -RRB- Hospital22Hospice33At home52025Other place112No -LRB- clear -RRB- preference16112231aInformation as provided by bereaved relative During the last 3 days of life , a large proportion of patients in both settings suffered , according to the reports of bereaved relatives , from fatigue , loss of appetite , pain , dyspnoea , and mouth or mucus problems -LRB- Table 4 -RRB- . Patients dying in an institution were somewhat more often incontinent and more often suffered from anxiety . Patients dying at home slightly more often had bedsores . There were no statistically significant differences between the settings in the degree to which symptoms were addressed with some form of medical treatment , except for loss of appetite , for which 6 of 22 patients who died in an institution received treatment , but only 2 of 30 patients who died at home -LRB- p = 0.04 -RRB- . The bereaved relatives ' evaluation of end-of-life care was in general positive in both settings . Some relatives of patients who died in an institution felt they had not sufficiently been involved in the decision-making , whereas all relatives in the other group were satisfied about their level of involvement . Table 4Dying in an institution or at home : perspective of bereaved relative Place of deathP value t testP value χ2 testIn institution -LRB- N = 29 -RRB- At home -LRB- N = 34 -RRB- Number of days patient stayed at place of death -LSB- mean -LRB- SD -RRB- -RSB- 7 -LRB- 6 -RRB- 59 -LRB- 35 -RRB- 0.000 Number of transfers -LSB- mean -LRB- SD -RRB- -RSB- 1.9 -LRB- 1.7 -RRB- 1.2 -LRB- 1.3 -RRB- 0.047 Number of disciplines involved with patient -LSB- mean -LRB- SD -RRB- -RSB- 6.1 -LRB- 2.3 -RRB- 5.8 -LRB- 1.9 -RRB- 0.64 Number of clinical specialties involved with patient -LSB- mean -LRB- SD -RRB- -RSB- 2.2 -LRB- 1.2 -RRB- 1.8 -LRB- 0.8 -RRB- 0.011 Patient -LSB- n -LRB- % -RRB- -RSB- Was fatigued22 -LRB- 79 -RRB- 29 -LRB- 88 -RRB- 0.33 Had loss of appetite22 -LRB- 76 -RRB- 30 -LRB- 88 -RRB- 0.20 Had pain20 -LRB- 69 -RRB- 29 -LRB- 85 -RRB- 0.12 Had dyspnoea18 -LRB- 62 -RRB- 17 -LRB- 52 -RRB- 0.40 Had mouth or mucous problems18 -LRB- 62 -RRB- 16 -LRB- 49 -RRB- 0.28 Was incontinent20 -LRB- 69 -RRB- 13 -LRB- 38 -RRB- 0.015 Had nausea9 -LRB- 32 -RRB- 10 -LRB- 30 -RRB- 0.88 Had bedsores5 -LRB- 17 -RRB- 13 -LRB- 38 -RRB- 0.07 Was confused14 -LRB- 48 -RRB- 10 -LRB- 29 -RRB- 0.12 Suffered from anxiety12 -LRB- 41 -RRB- 5 -LRB- 15 -RRB- 0.021 Was depressed3 -LRB- 10 -RRB- 2 -LRB- 6 -RRB- 0.54 Evaluation of care during the last 3 days of life -LSB- n -LRB- % -RRB- -RSB- Assistance with personal care was sufficient27 -LRB- 93 -RRB- 32 -LRB- 94 -RRB- 0.87 Nursing care was sufficienta26 -LRB- 90 -RRB- 33 -LRB- 97 -RRB- 0.23 Relative was involved in decision-making25 -LRB- 89 -RRB- 33 -LRB- 97 -RRB- 0.22 Relative was sufficiently involved in decision-making24 -LRB- 89 -RRB- 33 -LRB- 100 -RRB- 0.049 Patient might have disagreed with medical decision -LRB- s -RRB- 4 -LRB- 15 -RRB- 1 -LRB- 3 -RRB- 0.10 Relative disagreed with medical decision -LRB- s -RRB- 7 -LRB- 24 -RRB- 7 -LRB- 21 -RRB- 0.78 It had been clear that patient was dying18 -LRB- 67 -RRB- 26 -LRB- 79 -RRB- 0.29 aIncluding patients who did not need professional nursing care Further , of the bereaved relatives who had cared for a patient who died in an institution , 76 % reported that it had significantly affected their own social life ; this percentage was 94 % for relatives who had cared for a patient who died at home -LRB- Table 5 -RRB- . A substantial number of relatives had only received some or no support from other family members -LRB- 38 % -RRB- , and the number of relatives who reported that their own health had been affected by caring for the patient was also quite large -LRB- 38 % -RRB- . Caring for a dying relative rarely yielded financial problems . Nearly all relatives thought it very important that they had been involved in caring for the patient . Table 5Dying in an institution or at home : experiences of bereaved relatives Place of deathP value χ2 testIn institution -LRB- N = 29 -RRB- At home -LRB- N = 34 -RRB- n -LRB- % -RRB- How often did you see patient in the last months of life ? 0.78 Every day24 -LRB- 83 -RRB- 29 -LRB- 85 -RRB- Less than daily5 -LRB- 17 -RRB- 5 -LRB- 15 -RRB- Did caring for patient affect your own social life ? 0.068 Yes22 -LRB- 76 -RRB- 32 -LRB- 94 -RRB- No7 -LRB- 24 -RRB- 2 -LRB- 6 -RRB- Did caring for patient result in financial problems ? 0.17 Yes4 -LRB- 14 -RRB- 1 -LRB- 3 -RRB- No25 -LRB- 86 -RRB- 33 -LRB- 97 -RRB- Did you receive support from your family in caring for patient ? 1.0 Much support16 -LRB- 55 -RRB- 23 -LRB- 68 -RRB- Some or no support13 -LRB- 45 -RRB- 11 -LRB- 32 -RRB- Did caring for patient affect your own health ? 0.62 Yes12 -LRB- 41 -RRB- 12 -LRB- 35 -RRB- No17 -LRB- 59 -RRB- 22 -LRB- 65 -RRB- Did caring for patient cost you a lot of energy ? 0.96 Often or all the time15 -LRB- 52 -RRB- 15 -LRB- 44 -RRB- Rarely or sometimes14 -LRB- 48 -RRB- 19 -LRB- 56 -RRB- How important was being involved with care for patient for you ? 0.65 Very important26 -LRB- 90 -RRB- 32 -LRB- 94 -RRB- Important3 -LRB- 10 -RRB- 2 -LRB- 6 -RRB- Discussion Medical care for patients who are in the last stage of life has received much attention during the past decade in The Netherlands . The organization of health care services for terminally ill patients has greatly expanded during a 5-year period from 1998 through 2003 , during which the government financially supported six university-based centers for the further development of end-of-life care . After this period , the government took the position that , whereas death and dying are common events , the provision of end-of-life care should be part of the professional skills of all physicians , including general practitioners providing end-of-life care to patients who are staying at home . For complex problems , general practitioners and other physicians can now , in many places , seek support from expert teams -LSB- 20 -- 22 -RSB- . Of the 103 patients in this study , who were , at inclusion , all treated as outpatients by a clinical specialist , about half died in their own home . Death in an institution mostly concerned a hospital , which was probably not , for all patients , foreseen as the place of dying . Patient characteristics did not clearly determine the place of dying in our study . Whether or not patients lived with a partner was the only variable that was significantly associated with the place of dying . Having access to informal care support is invariably found to be a strong determinant of being able to die at home -LSB- 4 -RSB- . Other factors that have been found to affect rates of dying at home are the health status and emotional capacity of the main carer , the availability and use of home-based end-of-life care services , the need for specialist symptom control , tumor type , distance to inpatient services , gender and age of the patient , the patient 's socio-economic status , and strength and visibility of patient or carer preferences concerning the place and circumstances of dying -LSB- 4 , 23 -- 26 -RSB- . Obviously , the number of cases in our study was limited , and we did not assess all possibly relevant factors , which precludes firm conclusions on determinants of the place of dying . However , whereas access to the available public end-of-life care services , both institutional and home-based , is virtually unlimited in The Netherlands and financially covered by either private or public insurance , care needs of patients are probably a predominant factor . The availability of in - and outpatient end-of-life care services is probably another important determinant of place of death . The number of patients who died in a specialized end-of-life care service in our study was limited . National statistics on the place of dying does not include hospices or end-of-life care units as a separate category . However , the percentage of cancer deaths inside such services as found in our study is probably comparable to the percentage in the total Dutch population . Recently , the number of beds in specialized end-of-life care services in The Netherlands has been rapidly increasing -LSB- 27 -RSB- . It is , therefore , likely that the proportion of cancer deaths in end-of-life care services will further increase in the coming years . However , the extent to which dying in an end-of-life care service will substitute dying in a hospital or dying at home is hard to predict . Probably , institutional end-of-life care services address very diverse needs of dying patients and their caregivers . Further , financial and other incentives that are aimed at setting the course for the supply of end-of-life care services may affect developments concerning the place of dying and end-of-life care as well -LSB- 27 -RSB- . It is remarkable that a substantial number of patients in our study did not have a clear preference concerning their place of dying . Obviously , we only have information about the preferences that patients had discussed with their interviewed relative . Patients may also have had wishes that relatives were not aware of . Nevertheless , the data in our study suggest that the place of dying is not a great concern for a substantial number of terminally ill cancer patients in The Netherlands . In general , we found few differences in the evaluation by physicians and bereaved relatives of the dying phase of patients who died in an institution and patients who died at home . Patients who died in an institution were , according to their physicians , less often ready to die , which may be due to the fact that hospitalization is typically forgone in patients who are expected to die at short notice . A sudden and more rapid deterioration than expected may also have been among the reasons to admit patients to hospital shortly before dying in some cases . We did not find substantial differences between both settings in most end-of-life decision-making characteristics . Relatives in both settings quite often -LRB- 21 -- 24 % -RRB- stated that they had not agreed with the decision-making . Relatives of patients who died in an institution somewhat less often than other relatives felt that they had been sufficiently involved in the decision-making . Dissatisfaction of relatives with end-of-life decision-making in an institutional setting has been described elsewhere too -LSB- 28 -- 30 -RSB- and has been attributed to many factors , such as a lack of time of professional caregivers , lack of skills in communication , failure to recognize end-of-life decision-making as a subject that could be discussed , ethical barriers , and the lack of emotional support for relatives -LSB- 28 -- 32 -RSB- . It is unclear if our finding that relatives of patients who died outside an institutional setting more often feel satisfied about their involvement in the decision-making process is due to better communication in the home situation . However , the general practitioner , who often has a longstanding relationship with patients and their families , typically plays a key role in end-of-life care at home and may be better able to adequately communicate with family than institutional caregivers -LSB- 33 -RSB- . Medical decision-making may also be less complex or controversial for patients who die at home . The possibly rather complex decision-making in institutions is not associated with a higher prevalence of most symptoms . Fatigue , pain , and dyspnoea were among the most common symptoms in both settings , as has been found elsewhere too -LSB- 34 -RSB- . Incontinence was more common among patients dying in an institution , and this also holds for anxiety , which was reported as a problem for almost half of all patients dying in an institution . Incontinence and mental status have elsewhere also been found to be associated with a need of nursing home care -LSB- 35 -RSB- . In contrast , bedsores were more common at home . Virtually all relatives in both settings were satisfied about the personal and nursing care that had been provided to their dying relative . This may , to some extent , be indicative of the relative quality of end-of-life care in institutions and at home , but using satisfaction as an absolute indicator of the quality of palliative care services is quite problematic -LSB- 36 -RSB- . Further , most relatives were closely involved and appreciated their personal involvement very much , both for patients who died in an institution and patients who died at home . Nevertheless , caring for a dying relative often affected their own social life , especially when dying occurred at home , and took a lot of energy . Our study has several limitations . Firstly , the patients ' perspectives on their own dying process is lacking . Bereaved family members ' assessments are known to sometimes disagree with those of the patients ' , especially for subjective aspects such as psychological symptoms and pain -LSB- 37 -RSB- . This is also true for physicians ' assessment of their patients ' condition . Secondly , our group of patients is probably not representative for all patients dying from one of the five major types of cancer because patients with a very poor health status and patients who died shortly after the diagnosis of incurable cancer were not included in the cohort study upon which the data collection for this study was based . Moreover , data from bereaved relatives could only be obtained for 61 % of all patients , which may have resulted in an overestimation of the degree of satisfaction of relatives . The fact that patients of whom no bereaved relative participated in the study had a shorter disease duration at inclusion than other patients suggests that a rapidly progressive disease process makes it more difficult for bereaved relatives to talk about the last phase of life of the patient . In summary , we did not find major differences in the process and quality of dying between institutional settings and the home setting . Most patients with clear preferences concerning their place of dying were able to die at their preferred place . In about half of all cases , relatives were not aware of any preference of the patient concerning the place of dying , which is apparently not a major concern for many people . We conclude that the current situation in The Netherlands , in which the place of dying is mainly determined by the availability of informal caregivers at home and the care needs of the patient , involves no major threats to the process and quality of dying .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O

place of death
cancer patients
quality of dying


Downregulated parafibromin expression is a promising marker for pathogenesis , invasion , metastasis and prognosis of gastric carcinomas Parafibromin is a protein encoded by the hyperparathyroidism 2 oncosuppressor gene and its downregulated expression is involved in pathogenesis of parathyroid carcinomas . To clarify the roles of parafibromin expression in tumourigenesis and progression of gastric carcinomas , it was examined by immunohistochemistry -LRB- IHC -RRB- on tissue microarray containing gastric carcinomas -LRB- n = 508 -RRB- , adenomas -LRB- n = 45 -RRB- and gastritis -LRB- n = 49 -RRB- with a comparison of its expression with clinicopathological parametres of carcinomas . Gastric carcinoma cell lines -LRB- MKN28 , AGS , MKN45 , KATO-III and HGC-27 -RRB- were studied for parafibromin expression by IHC and western blot . Parafibromin expression was localised in the nucleus of gastric epithelial cells , adenoma , carcinoma cells and cell lines . Its expression was gradually decreased from gastritis to gastric carcinoma , through gastric adenomas -LRB- p < 0.05 -RRB- and inversely correlated with tumour size , depth of invasion , lymphatic invasion , lymph node metastasis and Union Internationale Contre le Cancer -LRB- UICC -RRB- staging -LRB- p < 0.05 -RRB- but not with sex or venous invasion -LRB- p > 0.05 -RRB- . Parafibromin was strongly expressed in older carcinoma patients compared with younger ones -LRB- p < 0.05 -RRB- . There was stronger positivity of parafibromin in intestinal-type than diffuse-type carcinomas -LRB- p < 0.05 -RRB- . Univariate analysis indicated cumulative survival rate of patients with positive parafibromin expression to be higher than without its expression -LRB- p < 0.05 -RRB- . Multivariate analysis showed that age , tumour size , depth of invasion , lymphatic invasion , lymph node metastasis , UICC staging and Lauren 's classification but not sex , venous invasion or parafibromin expression were independent prognostic factors for carcinomas -LRB- p < 0.05 -RRB- . Downregulated parafibromin expression possibly contributed to pathogenesis , growth , invasion and metastasis of gastric carcinomas . It was considered as a promising marker to indicate the aggressive behaviours and prognosis of gastric carcinomas . Introduction Parafibromin is a protein encoded by the hyperparathyroidism 2 -LRB- HRPT2 -RRB- oncosuppressor gene , whose mutation causes the hyperparathyroidism -- jaw tumour syndrome . The disease is an autosomal dominant disorder characterised by the occurrence of parathyroid adenoma or carcinoma , fibro-osseous jaw tumours of the mandible or maxilla and renal neoplastic and non-neoplastic abnormalities , such as Wilms ' tumour , hamartoma or cystic renal disease -LSB- 1 , 16 , 20 -RSB- . HRPT2 gene is located in human chromosome 1q31 .2 , consists of 17 exons and spans 18.5 kb in the genome . It encodes a 2.7-kb transcript which is translated into a 531-amino-acid parafibromin protein with a molecular weight of 60 kd -LSB- 3 , 4 , 23 -RSB- . The 200-amino-acid C-terminal segment of parafibromin shares 32 % identity and 54 % homology with cell division cycle 73 , a Saccharomyces cerevisiae protein forming the polymerase-associated factor 1 -LRB- Paf1 -RRB- complex , which is associated with ribonucleic acid -LRB- RNA -RRB- polymerase II and involved in transcript site selection , transcriptional elongation , histone H2B ubiquitination , histone H3 methylation , poly -LRB- A -RRB- length control and coupling of transcriptional and posttranscriptional events -LSB- 10 , 17 , 18 , 24 , 27 -RSB- . Parafibromin overexpression was documented to inhibit colony formation and cellular proliferation and induce cell cycle arrest in the G1 phase , indicating that parafibromin has a critical role in cell growth -LSB- 28 -RSB- . Northern blot analysis showed HRPT2 expression in heart , brain , placenta , lung , liver , skeletal muscle , kidney and pancreas -LSB- 4 -RSB- . Western blot study revealed parafibromin expression as a 60-kd band in the adrenal gland , heart , pancreas and kidney but 40-kd immunoreactive bands in the heart and skeletal muscle of human -LSB- 25 -RSB- . Immunohistochemically , higher expression of parafibromin was found widespread in glomerular mesangial cell , hepatocytes , cells of the base of gastric glands , renal cortex tubules and the pars intermedia of the hypophysis -LSB- 17 -RSB- . Subsequent investigations have revealed that mutations in HRPT2 are present in 66 -- 100 % of sporadic parathyroid carcinomas -LSB- 7 , 20 -RSB- . Hyperparathyroidism -- jaw-tumours-syndrome-related and sporadic parathyroid carcinomas are characterised by loss of parafibromin nuclear immunoreactivity -LSB- 5 , 22 -RSB- . Selvarajan et al. -LSB- 19 -RSB- found that parafibromin expression was inversely linked to tumour size , pathologic stage and lymphovascular invasion of breast carcinomas using immunohistochemistry in a tissue microarray -LRB- TMA -RRB- study . These findings suggested the potential roles of parafibromin in pathogenesis and progression of malignancies . Gastric carcinoma ranks as the world 's second leading cause of cancer mortality behind lung cancer despite a sharp worldwide decline in both its incidence and mortality since the second half of the 20th century -LSB- 9 -RSB- . Tumourigenesis and progression of gastric carcinoma is a multistage process with the involvement of a multifactorial aetiology , which mainly results from gene -- environment interactions -LSB- 32 , 33 -RSB- . Gastric carcinomas are classified into early and advanced ones on the basis of whether the carcinomas invade into the muscularis propria of the stomach -LSB- 12 -RSB- . In 1965 , Lauren -LSB- 13 -RSB- classified gastric carcinomas into intestinal - and diffuse-type ones based on the morphological appearances . Intestinal-type carcinomas are characterised by cohesive carcinoma cells forming gland-like tubular structures with expanding or infiltrative growth pattern . However , the cell cohesion is less apparent or absent in diffuse-type carcinoma and cancer cells diffusely spread in the gastric wall -LSB- 31 -RSB- . Generally , there is a favorable prognosis for the patients with early or intestinal carcinoma compared with the other type . In our study , parafibromin expression was examined in gastric carcinoma , adenoma , gastritis and gastric carcinoma cell lines and compared with the clinicopathological parametres of carcinomas , as well as prognosis to explore the clinicopathological significance and molecular roles of parafibromin expression in stepwise development of gastric carcinoma . Materials and methods Subjects Gastric carcinomas -LRB- n = 508 -RRB- were collected from the surgical resection , adenoma -LRB- n = 45 -RRB- from endoscopic biopsy or polypectomy and gastritis -LRB- n = 49 -RRB- from the endoscopic biopsy in our affiliated hospital , Himi Citizen Hospital and Kouseiren Takanoka Hospital between 1993 and 2006 . All carcinomas were adenocarcinomas and the adenoma group was free from non-neoplastic polyp types , leiomyomas and benign gastrointestinal stromal tumours . The patients with gastric carcinoma were 354 men and 154 women -LRB- 29 ~ 91 years , mean = 65.4 years -RRB- . Among them , 191 cases have carcinomas accompanied with lymph node metastasis . None of the patients underwent chemotherapy or radiotherapy before surgery . They all provided consent for use of tumour tissue for clinical research and our University Ethical Committee approved the research protocol . We followed up all patients by consulting their case documents or through telephone . Pathology All tissues were fixed in 4 % neutralised formaldehyde , embedded in paraffin and incised into 4-μm sections . These sections were stained by haematoxylin and eosin -LRB- HE -RRB- to confirm their histological diagnosis and other microscopic characteristics . The staging for each gastric carcinoma was evaluated according to the Union Internationale Contre le Cancer -LRB- UICC -RRB- system for the extent of tumour spread -LSB- 21 -RSB- . Histological architecture of gastric carcinoma was expressed in terms of Lauren 's -LSB- 13 , 31 -RSB- classification . Furthermore , tumour size , depth of invasion , lymphatic and venous invasion were determined . Tissue microarray Representative areas of solid tumours were identified in HE-stained sections of the selected tumour cases and a 2-mm-in-diametre tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer -LRB- AZUMAYA KIN-1 , Japan -RRB- . Four-micrometre-thick sections were consecutively incised from the recipient block and transferred to polylysine-coated glass slides . HE staining was performed on TMA for confirmation of tumour tissue . Cell lines and culture Gastric carcinoma cell lines come from the Japanese Physical and Chemical Institute , including MKN28 -LRB- well-differentiated adenocarcinoma -RRB- , AGS -LRB- moderately differentiated adenocarcinoma -RRB- , MNK45 -LRB- poorly differentiated adenocarcinoma -RRB- , KATO-III -LRB- poorly differentiated adenocarcinoma -RRB- and HGC-27 -LRB- undifferentiated adenocarcinoma -RRB- . They were maintained in Roswell Park Memorial Institute 1640 -LRB- MKN28 , MKN45 and KATO-III -RRB- , minimum essential -LRB- HGC-27 -RRB- or Ham 's F12 -LRB- AGS -RRB- medium supplemented with 10 % foetal bovine serum , 100-units/ml penicillin , and 100-μg/ml streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C . Total protein was prepared from all cells by cell disruption buffer according to Protein And RNA Isolation System manual -LRB- Arctiris Bioscience , USA -RRB- . All cells were collected by centrifugation , rinsed with phosphate-buffered saline , fixed by 10 % formalin and then embedded in paraffin as routinely processed . Immunohistochemistry Consecutive sections were deparaffinised with xylene , dehydrated with alcohol and subjected to antigen retrieval by irradiating in target retrieval solution citrate pH 6.0 -LRB- TRS , DAKO , Carpinteria , CA 93013 , USA -RRB- for 15 min with microwave oven -LRB- Oriental Rotor Lmt . Co. , Tokyo , Japan -RRB- . Five percent bovine serum albumin was then applied for 1 min to prevent non-specific binding . The sections were incubated with mouse anti-parafibromin antibody -LRB- Clone 2H1 , SC-33638 , Santa Cruz , CA , USA ; 1:40 -RRB- for 15 min , then treated with the anti-mouse Envison-PO -LRB- DAKO , CA , USA -RRB- antibody for 15 min . Binding sites were visualised with 3 , 3 ′ - diaminobenzidine with the 5-min reaction . All the incubations were performed in a microwave oven to allow intermittent irradiation as described previously -LSB- 11 -RSB- . After each treatment , the slides were washed with Tris-buffered saline with Tween 20 -LRB- TBST ; 10 mM Tris-HCl , 150 mM NaCl , 0.1 % Tween 20 -RRB- three times for 1 min . After being counterstained with Mayer 's haematoxylin , the sections were dehydrated , cleared and mounted . Omission of the primary antibody was used as a negative control . One hundred cells were randomly selected and counted from five representative fields of each section blindly by three independent observers -LRB- Takano Y , Li XH and Zheng HC -RRB- . The positive percentage of counted cells was graded semi-quantitatively according to a four-tier scoring system : negative -LRB- − -RRB- , 0 ~ 5 % ; weakly positive -LRB- + -RRB- , 6 ~ 25 % ; moderately positive -LRB- + + -RRB- , 26 ~ 50 % ; and strongly positive -LRB- + + + -RRB- , 51 ~ 100 % . Western blot Fifty-microgramme denatured protein was separated on an SDS-polyacrylamide gel -LRB- 10 % acrylamide -RRB- and transferred to Hybond membrane -LRB- Amersham , Germany -RRB- , which was then blocked overnight in 5 % milk in TBST . For immunoblotting , the membrane was incubated for 1 h with mouse anti-parafibromin antibody as described above . Then , it was rinsed by TBST and incubated with anti-mouse immunoglobulin G conjugated to horseradish peroxidase -LRB- DAKO , CA , USA , 1:1,000 -RRB- for 1 h. Bands were visualised with X-ray film -LRB- Fujifilm , Japan -RRB- by ECL-Plus detection reagents -LRB- Amersham , Germany -RRB- . After that , membrane was washed with WB Stripping Solution -LRB- pH 2 -- 3 , Nacalai , Tokyo , Japan -RRB- for 30 min and treated as described above except mouse anti-β-actin antibody -LRB- Sigma , MO , USA , 1:5,000 -RRB- as an internal control . Statistical analysis Statistical evaluation was performed using Spearman correlation test to analyse the rank data . Kaplan -- Meier survival plots were generated and comparisons between survival curves were made with the log -- rank statistic . The Cox 's proportional hazards model was employed for multivariate analysis . P < 0.05 was considered as statistically significant . SPSS 10.0 software was employed to analyse all data . Results Parafibromin expression in gastric tumours and carcinoma cell lines As shown in Fig. 1 , parafibromin was positively immunostained in the nucleus of MKN28 , AGS , MKN45 , KATO-III and HGC-27 , and its expression level was consistent with the data of Western blot . Parafibromin was strongly expressed in the nucleus of gastric epithelial cells , adenomas and early carcinomas but not in given advanced carcinomas . Occasionally , it also appeared in stromal fibroblasts and lymphocytes but much weaker than epithelial cells or adenomas -LRB- Fig. 2 -RRB- . Generally , the stromal lymphocytes and fibroblasts were negative in cases where the tumour was negative . Overall , parafibromin expression was detected respectively in all gastritis -LRB- 100.0 % -RRB- , 36 out of 45 adenoma patients -LRB- 80.0 % -RRB- and 233 out of total 508 gastric carcinoma patients -LRB- 45.9 % -RRB- . Statistically , gradually reduced expression of parafibromin was seen from gastritis to gastric carcinoma through gastric adenoma -LRB- p < 0.05 , Table 1 -RRB- . As summarised in Table 2 , parafibromin expression was inversely correlated with tumour size , depth of invasion , lymphatic invasion , lymph node metastasis and UICC staging -LRB- p < 0.05 -RRB- but not with sex or venous invasion -LRB- p > 0.05 -RRB- . Parafibromin was strongly expressed in older carcinoma patients compared with younger ones -LRB- p < 0.05 -RRB- . Intestinal-type carcinomas exhibited more frequent expression of parafibromin than diffuse-type ones -LRB- p < 0.05 -RRB- . Fig. 1Parafibromin expression in gastric carcinoma cell lines . a Parafibromin was positively immunostained in the nucleus of MKN28 -LRB- a -RRB- , AGS -LRB- b -RRB- , MKN45 -LRB- c -RRB- , KATO-III -LRB- d -RRB- and HGC-27 -LRB- e -RRB- . b Cell lysate -LRB- 50 µg -RRB- was loaded and probed with anti-human parafibromin antibody -LRB- 60 kd -RRB- with β-actin -LRB- 42 kd -RRB- as an internal control . Lane # 1 : MKN28 ; # 2 AGS ; # 3 MKN45 ; # 4 KATO-III ; # 5 HGC-27Fig . 2Immunohistochemical staining of parafibromin in gastritis , adenoma and carcinoma . Note parafibromin positivity was strongly observed in the nucleus of gastric superficial epithelium -LRB- a -RRB- , and adenoma -LRB- c -RRB- and early gastric carcinoma -LRB- b -RRB- , occasionally weaker in the stromal fibroblasts and lymphocytes -LRB- a , c -RRB- , but not in given advanced gastric carcinomas -LRB- d -RRB- , indicating that the internal positive control -LRB- stromal cells -RRB- was negative adjacent to the negative staining carcinoma cells but positive adjacent to the positive epithelial cellsTable 1Parafibromin expression in gastric tissue samplesGroupsNumberParafibromin expression − + + + + + + PR -LRB- % -RRB- Gastritis4901741100 .0 aGastric adenoma459582380 .0 bGastric carcinoma508275556011845 .9 PR Positive rateaCompared with gastric adenoma or carcinoma , p < 0.001 bCompared with gastric carcinoma , p < 0.001 Table 2 Relationship between parafibromin expression and clinicopathological features of gastric carcinomasClinicopathological featuresNumberParafibromin expression − + + + + + + PR -LRB- % -RRB- Rsp valueAge -LRB- years -RRB- < 6520912524243640.20.095 < 0.05 ≥ 6529915031368249.8 Sex Male35418837389146 .90.054 > 0.05 Female1548718222743 .5 Tumour size -LRB- cm -RRB- < 426311626348555.9 − 0.237 < 0.001 ≥ 424515927263335.1 Depth of invasion Tis − 126310230409161.2 − 0.344 < 0.001 T2-424517325202729.4 Lymphatic invasion − 33115742458752.6 − 0.168 < 0.001 +17711813153133.3 Venous invasion − 443236495110746.7 − 0.051 > 0.05 +6539691140.0 Lymph node metastasis − 31713838449756.5 − 0.285 < 0.001 +19113717162128.3 UICC staging 0 -- I29212336419257 .9 − 0.292 < 0.001 II-IV21615219192629.6 Lauren 's classification Intestinal-type27310834389360.4 − 0.322 < 0.001 Diffuse-type22515721222530.2 PR Positive rate , Tis carcinoma in situ , T1 lamina propria and submucosa , T2 muscularis propria and subserosa , T3 exposure to serosa , T4 invasion into serosa , UICC Union Internationale Contre le Cancer Univariate and multivariate survival analysis Follow-up information was available on 508 gastric carcinoma patients for periods ranging from 0.2 months to 12.2 years -LRB- median = 67.2 months -RRB- . Figure 3 showed survival curves stratified according to parafibromin expression for gastric carcinomas . Univariate analysis using the Kaplan -- Meier method indicated cumulative survival rate of patients with weak , moderate or strong parafibromin expression to be obviously higher than without its expression -LRB- p < 0.05 ; Fig. 3a -RRB- . The significant difference disappeared if stratified according to the depth of invasion -LRB- Fig. 3b , c -RRB- . Multivariate analysis using Cox ' s proportional hazard model indicated that age , tumour size , depth of invasion , lymphatic invasion , lymph node metastasis , UICC staging and Lauren 's classification -LRB- p < 0.05 -RRB- but not sex , venous invasion or parafibromin expression were independent prognostic factors for overall gastric carcinomas -LRB- p > 0.05 ; Table 3 -RRB- . Fig. 3Correlation between parafibromin status and prognosis of the gastric carcinoma patients . Kaplan -- Meier curves for cumulative survival rate of patients with gastric carcinomas according to the parafibromin expression in overall -LRB- a -RRB- , early -LRB- b , EGC -RRB- and advanced -LRB- c , AGC -RRB- gastric carcinomasTable 3Multivariate analysis of clinicopathological variables for survival with gastric carcinomasNumberClinicopathological parametresRelative risk -LRB- 95 % CI -RRB- p valueAAge -LRB- ≥ 65 years -RRB- 1.929 -LRB- 1.357 -- 2.743 -RRB- < 0.001 BSex -LRB- female -RRB- 1.463 -LRB- 0.983 -- 2.179 -RRB- > 0.05 CTumour size -LRB- ≥ 4 cm -RRB- 1.606 -LRB- 1.013 -- 2.547 -RRB- < 0.05 DDepth of invasion -LRB- T2 -- 4 -RRB- 6.530 -LRB- 3.110 -- 13.710 -RRB- < 0.001 ELymphatic invasion -LRB- + -RRB- 2.626 -LRB- 1.516 -- 3.374 -RRB- < 0.001 FVenous invasion -LRB- + -RRB- 0.959 -LRB- 0.633 -- 1.452 -RRB- > 0.05 GLymph node metastasis -LRB- + -RRB- 2.773 -LRB- 1.525 -- 5.043 -RRB- < 0.01 HUICC staging -LRB- II-IV -RRB- 0.294 -LRB- 0.139 -- 0.622 -RRB- < 0.01 ILauren 's classification -LRB- diffuse-type -RRB- 1.796 -LRB- 1.212 -- 2.661 -RRB- < 0.01 JParafibromin expression -LRB- + ~ + + + -RRB- 0.792 -LRB- 0.529 -- 1.187 -RRB- > 0.05 CI Confidence interval , UICC = Union Internationale Contre le Cancer Discussion HRPT2 has been isolated from complementary DNA libraries of parathyroid , kidney and bone tissue and encodes tumour suppressor protein parafibromin -LSB- 4 -RSB- . In the present study , the nuclear expression pattern was observed in the gastric epithelial cells , adenomas , adenocarcinomas and carcinoma cell lines consistent with previous reports in the gastric superficial mucosa , hepatocytes , kidney cortex tubules , adrenal gland , spleen lymphocytes , parathyroid tissue , adenoma and carcinomas , breast carcinoma -LSB- 5 , 8 , 17 , 19 -RSB- . Although the result was in contrast with the paper of Porzionato et al. -LSB- 17 -RSB- possibly due to different incubation times of primary antibody , the great majority of immunohistochemical and cell transfection studies supported our observation of the nuclear staining -LSB- 2 , 5 , 6 , 8 , 14 , 19 -RSB- . This study again demonstrates that parafibromin is nuclear and not cytoplasmic or nucleocytoplasmic in location as initially thought . The weaker expression of parafibromin in stromal cells than epithelial cells and adenoma might be due to the specificity of its cellular distribution as described previously -LSB- 17 -RSB- . It was found that the translocation of parafibromin to the nuclear compartment involved a function monopartite nuclear localisation signal at residues 136 -- 139 -LSB- 2 , 6 -RSB- . Parafibromin is a component of Paf1 complex in the nucleus , where it plays a role in cell cycle regulation , histone methylation , lipid and nucleic acid metabolism -LSB- 10 , 17 -RSB- . The distribution pattern of parafibromin protein in gastric epithelial cells or tumour cells demonstrated its biological function in the nucleus . Statistically , parafibromin expression was gradually reduced from gastritis to carcinoma through adenoma in line with parathyroid carcinogenesis , suggesting that downregulated parafibromin expression might contribute to the malignant transformation of gastric epithelial cells as an early event . The positive rate of parafibromin expression was reduced to 80 % in gastric adenoma and reached about 46 % of gastric adenocarcinoma , supporting the involvement of parafibromin in the gastric adenoma -- adenocarcinoma sequence . Actually , the adenoma can progress into and be incorporated with gastric well-differentiated carcinoma when it grows bigger and de novo carcinogenesis is well understood , especially in diffuse-type gastric carcinomas -LSB- 34 -RSB- . Higher parafibromin expression in adenoma and intestinal-type carcinoma indicated that decreased parafibromin expression might play an important role in de novo diffuse-type carcinogenesis but less in intestinal carcinogenic pathway . A body of evidences indicated that downregulation of tumour suppressor protein expression was due to genetic or epigenetic changes , like allelic loss , mutation , loss of heterozygosity -LRB- LOH -RRB- , hypermethylation and microsatellite instability in malignancies -LSB- 29 , 30 -RSB- . In the sporadic parathyroid carcinomas and hyperparathyroidism -- jaw tumours , LOH or mutation of HRPT2 might cause the loss and inactivation of parafibromin protein -LSB- 20 , 25 , 26 -RSB- . Furthermore , the reduced expression of parafibromin was found to closely link to the tumour size , depth of invasion , lymphatic or venous invasion and UICC staging in line with the observation in breast carcinomas -LSB- 19 -RSB- , indicating the inhibitory effects of parafibromin on tumour growth , invasion , metastasis and progression of gastric carcinomas . Drosophila Hyrax and its human orthologue , parafibromin , are required for nuclear transduction of the Wnt/Wg signal and bind directly to the C-terminal region of beta-catenin -- Armadillo , thereby controlling transcriptional initiation and elongation by RNA polymerase II -LSB- 15 -RSB- . Parafibromin overexpression can inhibit colony formation , anchorage-dependent cell growth and cellular proliferation and induce cell cycle arrest in the G1 phase -LSB- 28 -RSB- . These findings demonstrated that loss of parafibromin expression had impact on the pathogenesis and progression of malignancies by promoting cellular proliferation . Additionally , parafibromin was expressed with a higher incidence in intestinal-type gastric cancer , which is presumed to arise from preceding dysplastic lesions , than diffuse-type one , which evolves without any precedent dysplastic changes . It is also demonstrated that distinct parafibromin expression underlies the molecular mechanisms for the differentiation of intestinal - and diffuse-type carcinomas . Until now , there is yet no paper describing the prognostic significance of parafibromin expression in malignancies . Here , for the first time , we analysed the relation of parafibromin expression with the survival rate of 508 patients with gastric carcinoma . The results revealed a close link between its loss and worse survival . If stratified according to the depth of invasion , the significant link disappeared , indicating that the relationship between parafibromin expression and prognosis depends on the depth of invasion . The multivariate analysis demonstrated that age , depth of invasion , lymphatic invasion , lymph node metastasis , UICC staging and Lauren 's classification but not parafibromin expression , venous invasion or sex were independent prognostic factors for carcinomas . These findings suggested that parafibromin expression is a promising indicator for the favorable prognosis of gastric carcinoma patients , albeit not independent . In the present study , a large number of gastric carcinoma cases were screened by TMA , which takes the advantages of high throughput , identical immunohistochemical conditions , and economy of samples , antibodies and time -LSB- 33 -RSB- . Although we used 2-mm-in-diametre needles , which are large enough to evaluate the morphological appearance and carefully selected representative regions with the reference of HE slides , it was difficult to avoid selection bias . Gill et al. -LSB- 5 -RSB- found that stronger parafibromin staining and more positive cells sometimes appeared at the edges of the parathyroid tumour than in the centre . This could be due to fixation methods , other processing issues or a biological phenomenon , for example tissue hypoxia in the centre of large tumours . Additionally , the collection of our samples -LRB- e.g. gastritis , adenoma and adenocarcinoma -RRB- respectively from the endoscopic biopsy , polypectomy , or surgical resection put forward their another possibility of selection bias because of different fixation and processing methods . Selvarajan et al. -LSB- 19 -RSB- found that parafibromin underexpression was correlated particularly with large tumour size which is in line with our finding . It was possible that weaker staining could be attributable to poor fixation properties in the centre of large tumours . Because tumour size is a key prognostic indicator , this artifact could explain the prognostic significance of parafibromin in gastric carcinomas . Therefore , it is a limitation of the present study not to separate the edge and centre of gastric carcinomas when establishing TMA . In the current study , the negatively staining carcinomas are associated with a negative internal control -LRB- stromal cells and lymphocytes -RRB- whereas the positive staining epithelium is adjacent to positive staining internal controls . Therefore , the negative staining of the carcinoma might be artificial , which should be considered as another limitation of the study . Our study might be mentioned as a preliminary experiment and the staining with original-size sections is an extensive work in the future using the gastric carcinoma samples , fixed and processed by the same approach . In summary , downregulated parafibromin expression might play an important role in malignant transformation of gastric epithelial cells . Its reduced expression was closely related to growth , invasion , metastasis and worse prognosis of gastric carcinomas . Its expression could be employed to differentiate the intestinal - and diffuse-type carcinomas and underlay the molecular mechanism about the differentiation of both carcinomas . It was considered as a promising marker to indicate the pathobiological behaviours and prognosis of gastric carcinomas .

O B O O O O O O B O O O O O B O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O B O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O B O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O

gastric carcinoma
parafibromin
pathogenesis
progression
prognosis