This study investigated a) the amount and types of out-of-pocket expenditures by patients for nominally free services in a large public hospital in Bangladesh, b) the factors influencing these expenses, and c) the impact of these expenses on household income.Eighty-one maternity patients were interviewed during their hospitalization in the Dhaka Medical College Hospital. Patients were selected by quota sample to match the distribution of maternity patient categories in the hospital. Patients were interviewed with a semi-structured, in-depth questionnaire.All interviewees incurred substantial out-of-pocket expenditures for travel, hospital admission fees, medicine, tests, food, and tips. Only two of the expenditures, travel expenses and admission fees, were not supposed to be provided free of charge by the hospital. The median total per-patient expenditure was $65 (range $2–$350), equivalent to 7% (range 0.04%–225%) of annual household income. Half of all patients reported that their families had to borrow to pay for care at interest rates of 5%–30% per month. A third of these families reported selling jewelry, land or household items to moneylenders. The rural patients reported more difficulty in paying for care than the urban patients. Factors increasing the expenditures were duration of hospitalization, rural residence, and necessary (e.g. C-section, hysterectomy) and unnecessary (e.g. episiotomy) medical procedures.Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities. In developing countries governments often subsidize services at public health care facilities and provide them free of charge to users. However, evidence suggests that users still incur large expenditures using the 'free' services for such things that are supposedly provided without charge. Studies have found that patients incurred substantial out-of-pocket expenditures for medicine, food and travel from the use of 'free' public health facilities -3. A stuThree previous studies have explored issues related to patient expenditures in Bangladesh ,10,11. NThus, this study examined the type, amount and household financial results of out-of-pocket expenditures by patients for nominally free services in a large government hospital in Dhaka. The study also identified the factors and medical practices producing and influencing the out-of-pocket expenditures. Plans to begin fees for service in Bangladesh make it important to document the amount of money actually being paid by the patients under the present system. If current expenditures are large, fee for service may have serious negative impacts on utilization and on the economic well-being of Bangladeshi households. If current expenditures are modest, it is possible that such fees will have a lesser impact.The study was conducted in the Department of Obstetrics and Gynaecology (ObGyn) of the Dhaka Medical College Hospital (DMCH). DMCH is the largest teaching hospital in Bangladesh with 850 beds located in the capital city. DMCH is government funded and provides a wide range of out- and in-patient services. Public hospitals have two payment categories for in-patients: non-paying and paying. Patients first go to an out-patient unit for diagnosis where they are categorized as out- or in-patient. Those categorized as in-patient are then classified as paying or non-paying by observing the clothes and general appearance of the woman and any accompanying relatives. Non-paying patients pay only the hospital admission fee. Paying in-patients are charged the fees for hospital admission, bed, and surgery. The various fees are: hospital admission fee: $0.23, bed fee: $1.34–3.50 per day, surgery fee: $12.50–125. Taka was converted into US dollars using the 1994 exchange rate of US$1.00 = Taka 40.00. Neither patient category is supposed to pay for medicine, tests, food, nursing and other support services during hospitalization; these commodities and services are theoretically provided free by the hospital.The study interviewed 81 non-paying in-patients hospitalized for reproductive health conditions (about two thirds were for maternity conditions). Patients were selected by quota sample matching the distribution of the patient categories in the hospital i.e. the selected medical conditions accounted for the greatest number of ObGyn admissions reported for the hospital, and also reflect the causes associated with high maternal mortality and morbidity in Bangladesh . These iInformation was collected on various characteristics of the study participants. Demographic characteristics included age, education, marital status, and residence. Socio-economic characteristics included occupation and annual household income. Information was also collected on underlying medical condition. Out-of-pocket expenditure related information included types and amounts of expenses incurred during hospitalization such as those for travel, medicine, food, fees, etc. Factors influencing expenditures included type of treatment received and duration of hospitalization. Sources of funds included amount borrowed and interest charged for borrowed amount.Data were collected from patients and their relatives with semi-structured open-ended questionnaires between January – June 1994. The interviewers were physicians employed in DMCH. The interviewers selected the cases by diagnosis from patient admission records. To minimize possible selection bias the first case was selected randomly from the records and then every third case was selected. The selected patients were interviewed a minimum of three times to minimize recall error. Recall error was also minimized as information was collected while patients were still hospitalized. During the first interview demographic and socio-economic information was collected with structured questions. During the second and third interviews information related to expenditures was collected with open-ended questions.To illustrate the data collection process a description of an interview with a typical C-section patient follows. C-section patients are usually hospitalized for two weeks in DMCH. On the first day of hospitalization an interviewer collected information on patient's age, education, marital status, etc. On the eighth day of hospitalization the second interview collected information on treatment received, treatment related out-of pocket expenditures, annual household income, amount of money borrowed to pay for treatment, source of borrowed money, and interest rate charged. On the fourteenth day the third interview collected more monetary information on out-of-pocket expenditures, and on expected expenditures immediately after leaving the hospital. This survey did not cover the expenditures for the full course of the treatment. Expenditure estimates were derived for the duration of the current hospitalization only, i.e. from the day of admission until the day of discharge. Expenditures immediately before admission and after discharge from the hospital included only travel expenses to and from the hospital for the patient and her accompanying relatives.The median age of the study participants was 26 years (range 15–60 years). The majority (88%) of the patients were married, the rest were separated (4%), divorced (2%), and unmarried (1%). Forty-four percent of the patients lived in rural areas. The median annual household income was $750 (range $3–$6000) per respondent. The annual household income was higher for the urban  than the rural  respondents.All 81 patients interviewed reported incurring substantial out-of-pocket expenditures during their hospitalization. These out-of-pocket expenditures were for travel, hospital admission fee, medicine, tests, food, tips, and other items. As expected there were expenditures related to travel and admission fees which the hospital is not supposed to subsidize. But there were also expenditures for medicine, tests, food, tips, and other items which were supposed to be provided free from the hospital but were not.The median total expenditure for hospitalization was $65 range $2.15–$350) per patient. On average, 61% of these expenditures ($49) were for services and commodities that were supposed to be provided free from the hospital but were not. The per patient median expenditure for the various expense categories were: medicine $26, tests 0, tips $1.25, food $1.25, other items $4.38, travel $22.25, and hospital admission fee $0.25. On average, medicine constituted 42%, travel 38%, tests 5%, food 4%, tips 2%, admission fees <1%, and others 8% of the total expenditures. C-section and hysterectomy cases had the highest median expenditures. Table .15–$350 All patients were supposed to be provided required medicines free from the hospital but were not. Medicines included antibiotics, analgesics, syringe, catheter, blood, and so forth. Medicine was usually bought when patients were admitted at night. The medicine required for treatment is ordered by the on-duty physician but it takes several hours for the hospital management to process the order. Thus, no free medicine is available immediately. To start the treatment, the on-duty physician requests the patient's relatives to buy the medicine which is purchased from nearby private pharmacies.All tests (e.g. pathology, radiology) are supposed to be provided by the hospital but sometimes the patients had the tests done in a private laboratory because waiting time for tests is very long in the DMCH due to the high patient load.ayahs (cleaning ladies) or nurses to provide necessary services. Thus, food was usually bought from a vendor or brought from home for both patient and relatives.Food is provided by the hospital but the interviewees found the hospital food of poor quality or totally lacking . Relatives usually stayed with the patient in the hospital because of lack of bakshish) are payments made to ayahs and guards. Ayahs were given tips for routine services such as pushing the patient's trolley to and from the labour/operation room, shaving the patient before delivery/surgery, giving enemas, etc. Guards at the gates were tipped each time a relative came to visit the patient during non-visitor hours. However, ayahs and guards are salaried hospital employees and are supposed to provide these services free of charge. The patients were reluctant when talking about the tips probably because they were still hospitalized and depended on these employees for access to certain services.Tips (The other expenditures included items for the patient (e.g. hot water, bucket for hot water) and the newborn baby (e.g. blanket) that were supposed to be provided by the hospital free of charge but were not.Travel expenses are not supposed to be provided by the hospital. Travel expenses consisted of travel to and from the hospital by the patient and any accompanying relatives, and travel expenditures of relatives during hospitalization for purchasing medicine and food for the patient. The patients came to DMCH because they expected 'free' and 'affordable' services compared to private clinics, or they were referred from a primary/secondary level facility, or to get better treatment here. Patients from rural/peri-urban areas took longer to reach DMCH than those from urban Dhaka .This expense is also not supposed to be covered by the hospital. The official price of admission was $0.23, but it was zero for two patients and more than the official price for half of the patients interviewed. The study could not elicit the reason the patients paid more than the official price. When probed the patients could not or would not elaborate beyond the amount paid. The patients were very reluctant when talking about paying more than the official price for the admission fee.Duration of hospitalization and rural residence of the patients increased the out-of-pocket expenditures. Rural residence increased the travel expenses and thus the total expenditures. Longer duration of hospitalization increased virtually all expenditures. The median duration of hospitalization was 8 days (range 1–34 days) per patient. Duration of hospitalization was the longest for hysterectomies followed by C-sections. Duration of hospitalization was related to severity of medical condition (e.g. eclampsia), necessary medical procedures (e.g. hysterectomy), and unnecessary medical procedures (e.g. episiotomy). One day of extra hospitalization increased expenditures by $2.30 per patient.medical procedures increased the patient expenditures. Episiotomy increased expenditures as patients were hospitalized for a longer duration and resulted in the purchase of more medicine. Episiotomy increased expenditures for both uncomplicated NVD (by 37%) and eclamptic NVD (by 84%) compared to cases where no episiotomy was performed (data not shown). The medical reason for performing episiotomies is the prevention of perineal tearing but because of a high case load at DMCH physicians perform episiotomies to reduce the length of delivery time, effectively turning hospital expenditures into patient expenditures. Eclampsia increased the expenditures for NVD by 180% (data not shown). Eclampsia is not under the control of the health system or the patient, and procedures used for treating eclampsia are unavoidable.Choice of C-sections caused higher patient expenditures compared to NVDs (median $119 and $63 respectively) because C-sections had a longer duration of hospitalization and required more medicine. Elective C-sections and eclamptic C-sections incurred similar expenses because elective C-sections were hospitalized for a longer duration even though there were no complications. Vaginal hysterectomies were 25% less expensive than abdominal hysterectomies because they required less invasive procedures, used local anaesthesia, and had a shorter duration of hospitalization.The respondents said that they were willing to pay for care. However, rural households reported more difficulty in paying for care than urban households. Difficulty was inferred from the number of households who borrowed to pay for care, and the ratio of the amount borrowed to the annual household income. The median patient expenditure was equivalent to 7% (range 0.04%–225%) of annual household income, and was higher for rural  than urban  respondents. Half (n = 40) of the households reported borrowing to pay for care. The patient who spent 225% of her annual household income was a rural patient who had a hysterectomy for prolapsed uterus. Surgical patients like her are usually hospitalized for a month as they require more tests than non-surgical patients. This patient's total expenditures were not higher than the others who also had a hysterectomy, however, her annual household income was much lower than that of the others.More urban n = 23) households borrowed than rural (n = 17) households but the amount borrowed was higher for rural households. The median amount borrowed per household was $38 and was equivalent to 8% (range 0.58%–208%) of the annual household income. On average, the rural households  borrowed almost double the amount than the urban households . Most often (n = 30) money was borrowed from friends and relatives without interest. When borrowing was from money-lenders (n = 10), households reported interest rates of 5%–30% per month. Three households put up security such as jewelry, land and household goods when borrowing money from moneylenders. The highest reported percentage of money borrowed to income for a rural household was 208% compared to 28% for an urban household. Finally, greater amounts of money were borrowed by C-section and hysterectomy patients than the other categories of patients. Table  householThe study findings indicate that all the surveyed patients incurred substantial out-of-pocket expenditures for a one time hospitalization. The median per patient expenditure was $65, and two-thirds of these expenditures were for commodities and services that were supposed to be provided free by the hospital but were not. Half the households borrowed to pay for care since they did not have the ability to pay . A third of these households sold jewelry, land or household items to moneylenders. The rural households reported more difficulty in paying for care than the urban households. The rural patients had lower income but incurred higher expenditures and borrowed larger amounts than the urban patients.The study data are a decade old but worth presenting because Bangladesh is only recently beginning health sector reform and fee for service. Also, the hospital practices with regard to providing maternity care remain the same, in the hospital studied and in other public hospitals in Bangladesh. Other limitations of the study are its small sample size and that all the interviewees came from only one hospital. The small sample size is the norm when doing in-depth interviews. When a paper is qualitative not quantitative no statistical tests are customarily done. The results are striking despite the limitations. The costs related to NVD and C-section were much higher in this study than that estimated by Nahar et al. . Possible reasons for the differences are: a) recall error higher in the Nahar et al. study as they interviewed post-partum mothers, whereas, this study interviewed patients during hospitalization; b) the Nahar et al. study included only uncomplicated cases, whereas, this study included both uncomplicated and complicated cases. The annual household income was lower in this study compared to the Nahar et al. study ($750 and $1476 respectively). This may be attributable to having rural patients in this study whose income is much lower than urban patients, whereas, Nahar et al. studied only urban patients.Changing current practices regarding the length of hospitalization and medical procedures (e.g. episiotomy) will reduce patient expenditures. This can be achieved by having shorter duration of hospitalization for elective C-section and elective hysterectomy, and limiting the use of episiotomy. Limiting these practices may also lead to lower provider expenditures. Medicine constituted almost half of the total patient expenditures. However, lack of access to medicine resulted from an inefficient management system not from unavailability. Taking less time to process orders would make medicine available quicker to patients and so reduce their expenditures.Hospital management needs to ensure that patients pay only the official rate of admission fees. Hospital management also needs to ensure that patients do not pay tips to salaried hospital staff for routine services. The hospital could arrange for liquid food and hot water for surgical patients in addition to the other services it already provides, i.e. provide a more comprehensive hotel service. Alternatively the hospital could subcontract out its hotel services to those employees who extract tips from patients to provide such services. This may act as an incentive for eliminating the unofficial fees from tips. Making some services such as eclampsia and hysterectomy available at secondary level facilities will benefit the rural patients by reducing the travel expenses. Government hospitals can generate revenue by introducing or increasing some fees for medicine and tests for the paying category of patients but clearly exempting the poorest. How would the system determine who was the poorest? One option would be to let all rural women use hospital for free and have all urban women pay some fee.For service fees not to become a serious barrier to use current systems will have to eliminate or reduce practices that already result in high user expenditures from unofficial payments. Otherwise the added fees may lead to more borrowing from moneylenders, putting lands and goods at risk, and potential impoverishment of more households. A fee for service system needs to be based on information, and more than just setting a price. This can be facilitated by knowing what the patients were already spending in the current system and who were the most at risk of impoverishment. The challenge is to focus on realistic, short term changes that can reduce patient expenditures and inequities. Most of the recommendations of this study depend more on the will of the physicians and the hospital administrators than on the infusion of resources per se.Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities.The author(s) declare that they have no competing interests.


Cost utility analysis (CUA) using SF-36/SF-12 data has been facilitated by the development of several preference-based algorithms. The purpose of this study was to illustrate how decision-making could be affected by the choice of preference-based algorithms for the SF-36 and SF-12, and provide some guidance on selecting an appropriate algorithm.Two sets of data were used: (1) a clinical trial of adult asthma patients; and (2) a longitudinal study of post-stroke patients. Incremental costs were assumed to be $2000 per year over standard treatment, and QALY gains realized over a 1-year period. Ten published algorithms were identified, denoted by first author: Brazier (SF-36), Brazier (SF-12), Shmueli, Fryback, Lundberg, Nichol, Franks , and Lawrence. Incremental cost-utility ratios (ICURs) for each algorithm, stated in dollars per quality-adjusted life year ($/QALY), were ranked and compared between datasets.In the asthma patients, estimated ICURs ranged from Lawrence's SF-12 algorithm at $30,769/QALY (95% CI: 26,316 to 36,697) to Brazier's SF-36 algorithm at $63,492/QALY (95% CI: 48,780 to 83,333). ICURs for the stroke cohort varied slightly more dramatically. The MEPS-based algorithm by Franks et al. provided the lowest ICUR at $27,972/QALY (95% CI: 20,942 to 41,667). The Fryback and Shmueli algorithms provided ICURs that were greater than $50,000/QALY and did not have confidence intervals that overlapped with most of the other algorithms. The ICUR-based ranking of algorithms was strongly correlated between the asthma and stroke datasets (r = 0.60).SF-36/SF-12 preference-based algorithms produced a wide range of ICURs that could potentially lead to different reimbursement decisions. Brazier's SF-36 and SF-12 algorithms have a strong methodological and theoretical basis and tended to generate relatively higher ICUR estimates, considerations that support a preference for these algorithms over the alternatives. The "second-generation" algorithms developed from scores mapped from other indirect preference-based measures tended to generate lower ICURs that would promote greater adoption of new technology. There remains a need for an SF-36/SF-12 preference-based algorithm based on the US general population that has strong theoretical and methodological foundations. Health-related quality of life (HRQL) measures have many applications, including the measurement of population health status and outcomes of medical interventions that subsequently can be applied to economic evaluations of health care interventions. One such method of economic evaluation, cost utility analysis (CUA), is a special form of cost effectiveness analysis that evaluates incremental costs and effects of an intervention by assessing health effects using quality-adjusted life years (QALYs) . QALYs iOptimally, CUA is used to guide the allocation of resources on a societal level. The Panel on Cost Effectiveness in Health and Medicine recommended that community preferences for health states collected from a representative sample of the US general population should be "the most appropriate ones for use in a Reference Case analysis" for US decision makers . Such anAn important development in health services research has been the emergence of algorithms that generate single preference-based summary scores based on items, domain scores, or summary scores from the Short Form 36 (SF-36)  and SF-1The purpose of this study was to examine how choice of algorithm for the SF-36/SF-12 might affect decision-making. The specific objectives for the study were to calculate ICURs by applying each algorithm to data from 2 different studies that included longitudinal assessments of the SF-36, to compare the ranking of each algorithm-based ICUR across conditions, and finally to interpret whether differences in ICURs generated by each algorithm had the potential to affect decision making. There were two specific hypotheses. First, ICURs calculated from different algorithms were expected to differ because preferences derived from those algorithms had been found to be different . Second,To illustrate the outcomes of CUA using the different SF-36 algorithms, data with empiric responses to the SF-36 from patients were used from two different sources and conditions: (1) a clinical trial of adults with asthma ; and (2)The second source of data was a natural history of HRQL after stroke. Stroke patients who were hospitalized with a confirmed ischemic stroke and consented to participate were included. Patients were excluded if they were ≤ 18 years old, could not comprehend English-based questionnaire, lived > 150 kilometers from Edmonton, Alberta, had hemorrhagic or lower brain stem stroke, coma, global or Wernicke's aphasia, or life expectancy was less than 6 months for any medical reason. Patients were enrolled in the study within two weeks of stroke and no later than 3 weeks after stroke. Health status measures, including the SF-36, were self-assessed by patients. For this analysis, responses to the SF-36 at baseline and 6 months were used. Both the stroke and asthma studies used version 1 of the SF-36.The SF-36 has been traditionally described as a psychometrically-derived generic health status profile, with 8 subscales and two summary scores, the physical component summary (PCS-36) score and the mental component summary (MCS-36) score. The eight domains include physical functioning (PF), role limitations-physical (RP), bodily pain (BP), general health (GH), mental health (MH), role limitations-emotional (RE), vitality (VT), and social functioning (SF). The SF-12 is a shorter, 12-item version of the SF-36 that does not generate domain scores but provides summary scores, the PCS-12 and MCS-12, that are highly predictive of the PCS-36 and MCS-36 . Scores st generation if the algorithm was derived from directly elicited preferences, and denoted as 2nd generation mapping if the SF-12/SF-36 algorithm was based upon scores from an indirect preference-based HRQL measure, such as the EQ-5D. Note that these algorithms relate to the most recently advocated algorithms, as several authors published earlier algorithms and subsequently published updates (e.g. Shmueli) [Nine publications that derived 10 unique preference-based algorithms for the SF-36 or SF-12 were identified (Table Shmueli) . For breBrazier and colleagues constructed an econometric model for predicting health state valuations by first revising the SF-36 into a health status measure with 6 domains called the SF-6D . Using aFryback and colleagues predicted Quality of Well-being Index (QWB) scores from SF-36 domain scores using data collected from the Beaver Dam Health Outcomes Study . A six-vNichol and colleagues mapped the SF-36 to the preference-based Health Utility Index Mark 2 (HUI2). They estimated HUI2 scores from SF-36 domain scores and sociodemographic variables from a sample of Southern California Kaiser Permanente members . The NicShmueli updated an examination of the relationship between Visual Analog Scale (VAS) ratings and SF-36 domains provided in a population health survey in Israel by predicting VAS values from SF-36 domains using linear and non-linear regression models . The modFranks and colleagues mapped the SF-12 to the EQ-5D Index and HUI3 using a convenience sample of 240 low-income, predominantly Latino and black patients visiting a community health center in New York . Two equFinally, Lundberg and colleagues investigated the relationship of preference-based measures and the SF-12 based on self-assessed HRQL from a random sample of residents in Uppsala County of Sweden . Linear Empiric data for stroke and asthma were used to ensure that actual health state changes were represented. The present analysis was based on patients who completed both pre- and post- assessments and had no missing items. After the scoring algorithms were applied to SF-36 responses using the 10 algorithms , the chat-tests. The rank order of the ICURs was compared between the asthma and stroke groups using Spearman's correlation coefficient (rs). P-values < 0.05 were considered statistically significant.The pre/post domain and preference-based scores were described for both study groups visit using means and standard deviations. The 95% confidence intervals (CIs) for ICURs were based on the CIs for the preference scores. The pre/post change scores were evaluated with paired Of the 304 patients enrolled in the asthma study, 220 (72.4%) completed both the baseline and final SF-36 assessment. The stroke study had 81 of 124 initial respondents (65.3%) complete the SF-36 at baseline and final follow-up. In comparison to the patients in the asthma study, patients in the stroke study were older (mean age 67.4 years vs. 39.1 years) and had much lower mean average PF, RP, SF, and PCS scores  Table . In the s = 0.60 .Table The development of preference-based algorithms for the SF-36 and SF-12 to facilitate CUA has fostered studies that recognized these preference-based scores can differ from each other and from directly elicited valuations in patients with asthma, hypertension, lung transplantation, and osteoporosis ,20,29,30Although guidelines or thresholds for decision making based on cost per QALY are contentious, cost-effectiveness thresholds that health care decision makers are willing to accept in health care reimbursement decisions exist, if not explicitly, then implicitly. Some guidance has been published. The National Institute of Clinical Effectiveness (NICE) in the UK has indicated they do not have an explicit threshold , while aThe CUA simulations illustrated how selection of a specific algorithm could lead to a different interpretation of the cost-effectiveness of an intervention. In the asthma cohort, algorithms by Lawrence , and theChanges in the rank order of algorithms between conditions can be explained, not only by differences in the preference-based weighting assigned to each of the domain/summary scores, but several additional factors. There are differences in the SF-36 items or subscales retained by some of the methods. For instance, Brazier's SF-6D based on the SF-36 does not include GH, while the score generated by the Shmueli algorithm is largely influenced by the GH domain. The responsiveness/sensitivity of algorithms appears to be somewhat related to the scale range. It was not surprising that Fryback's method produced relatively larger ICURs that implied the intervention was less cost effective, as Fryback's method had a much smaller range of scale relative to the other algorithms Table . AlgoritIn order to provide some guidance in the selection of preference-based algorithms for the SF-36 and SF-12 the algorithms were appraised in the context of their theoretical and methodological foundations, source of community-based preferences, and their relative potential to enhance or deter the uptake of new technology. The study results clearly illustrated that choice of algorithm can affect the estimated ICUR, and that there was a tendency for the Fryback and Shmueli methods to generate higher estimates of ICURs relative to the other algorithms. From a third-party payer perspective, algorithms generating higher ICURs would appeal to third-party reimbursement decision makers with short-term budget constraints, as algorithms that generate higher ICURs provide less encouraging evidence in the adoption of new technology when considered in the context of the $/QALY benchmarks previously discussed. The Panel on Cost Effectiveness of Health and Medicine . InteresA further consideration is the theoretical foundation for the elicitation technique used in the valuation study. Only Brazier employed methodology using the SG technique for first-generation mapping. The SG has the most appeal in economic theory due to its foundations in Expected Utility Theory (EUT), although it has been suggested that the axioms of EUT are empirically flawed , and reqGiven that HRQL may be valued differently between countries , an algoAmong the algorithms presently available, Brazier's algorithms for the SF-12 and SF-36 appear to be most favorable because of their methodological and theoretical basis. From the perspective of the third party reimbursement decision maker, the Brazier algorithms are not among those that tend to encourage adoption of new technology, tending to provide relatively higher estimates of ICUR. For those decision-makers, those algorithms would appear to be more fiscally conservative in the sense that they would not promote the adoption new technology any more than the other methods according to the results of this study. Although similar estimates are obtained using the Brazier SF-36 and SF-12 algorithms , it woulNote that there are several limitations and assumptions to the CUAs simulated in this paper. The primary purpose was to determine whether choice of preference-based algorithm applied to SF-36/SF-12 data has the potential to change the conclusion of a CUA; hence, aspects of the CUA not central to the purpose were simplified. For example, incremental costs were assumed to be constant, whereas in reality, considerable cost variance would be observed across patients. CUAs were performed in two patient populations, i.e., asthma patients and stroke patients, rather than using a single data set, to enhance the generalizability of the results. The rank order of the algorithms is limited to the datasets examined in this study, however, and comparisons of the algorithms across more diseases/conditions and persons with different demographic characteristics may provide stronger evidence of the rank order "stability". Change scores and thus ICUR estimates depend on baseline health status and the impact of an intervention on the various domains of health as captured by changes in responses to the SF-12/SF-36 items. Note that baseline domain scores for asthma and stroke cohorts were lower than US-based population norms. Several algorithm developers provide caveats for the application of their algorithm, including concerns about profiles severely limited by ceiling or floor effects . For insIn summary, SF-36/SF-12 preference-based algorithms tend to generate a wide range of ICURs that can potentially lead to different reimbursement decisions. Brazier's algorithms for the SF-36 and SF-12 had an arguably stronger methodological and theoretical basis, and tended to produce higher ICURs. For decision-makers who consider cost-effectiveness in the decision to reimburse for a new medical technology, selection of an algorithm that generates relatively higher ICURs would provide less convincing evidence of the cost-effectiveness of a new technology and consequently, its uptake. The "second-generation" algorithms mapped from other indirect preference-based measures tended to produce lower ICURs. When an alternative algorithm is selected, sensitivity analysis is recommended using the Brazier SF-12/SF-36 algorithm in order to examine the robustness of CUA. There remains a need for an SF-36/SF-12 algorithm developed from U.S.-based general population preferences with strong methodological and theoretical foundations.Drs. Pickard and Lee were responsible for the conception of the study and acquisition of data. Mr. Wang analyzed the data. Dr. Pickard and Mr. Wang were involved in the drafting of the article. All authors contributed to the interpretation of results and revising the article for important intellectual content.


Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of  cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading:. I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues  from three non-astrocytoma patients were included as the control.ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVINABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues . Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.Seventeen genes (Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma. EGFR) [INK4A initiated cell-cycle arrest pathway (the p16INK4a) [p14ARF [RB1 [the p53 [CD44, integrin, and receptors for various growth factors), and the PTEN genes [Diffusely infiltrating astrocytoma is a leading group of the primary central nervous system tumors, accounting for more than 60% of all primary brain tumors ,2. It maEGFR) , the mai [p14ARF , the RB1[the p53 ), a wideEN genes .de novo DNA methylation in the early development of high eukaryotes. DNA methylation patterns in somatic cells are established during the early development and contribute to the allele-specific transcription silencing of the imprinted genes, including the silenced alleles in the X-chromosome and other chromosomes. The epigenetic pattern (the DNA methylation profiles of the genome) in high eukaryotes is integral to the normal execution of the biological activities in cells and needs to be actively maintained. In addition to the changes linked to the cell lineage specific pattern of gene expression, both global hypomethylation and local hypermethylation of the CpG islands occur progressively as cell ages.Transcription in eukaryotes is regulated at multiple levels and inversely correlated with the hypermethylated state as well as the chromatin condensation. It has been well established that the methylation status of CpG islands directly affects the DNA-protein interactions by eliminating the otherwise occurring sequence specific binding of the transcription factors whereas inducing the DNA-bindings of members of the methyl-CpG binding protein family (MBD). Histone modifications (deacetylation and methylation) may occur subsequently leading to chromatin condensation and a long-term transcriptional silencing status of the affected DNA segments. Over 40% of the protein coding genes have at least one CpG island within or near to their promoter, an strong indication for transcription of which is likely to be under the control of DNA methylation status. Three DNA methyl transferases are involved in the control of the methylation state of the CpGs in genome. DNA methyl transferase I is mainly responsible for the maintenance of the methylation status of the genome after DNA replication, while IIIA and IIIB act principally in the p16INK4a gene that has been frequently hypermethylated in almost all types of the tumors examined [SURVIVIN, has also been reported to be demethylated in human ovarian cancer [Aberrant DNA methylation pattern changes gene transcription that has been etiologically linked to cancer formation ,10. The examined -17 incluexamined . The losexamined . On the examined . A promiexamined , correlaexamined ,22. The n cancer . Despite. However, as the majority of studies had only targeted one or a few genes in rather small patient groups, the concurrent hypermethylation behavior of multiple genes has only been addressed in a limited number of tumor types, such as colorectal cancer. The majority, if not all, of the previous studies on the astrocytoma associated changes in methylation profiles have only examined a small number of genes for methylation status at the promoter CpG island [Methylation profiling of the promoter CpG islands has been an important information gathering process for new insights into our understanding of the role of DNA methylation in both initiation and progression of human carcinogenesis. It would result in development of the DNA methylation based assays for cancer diagnosis as well as identification of the cancer genes suffering from the epigenetic defects G island ,26. In tARF, p15INK4b, RASSF1A, RB1 APC, BRCA1, CDH1, DAPK1, hMLH1, p14and VHL) or designed according to the same principle with assistance of the software packages for the CpG islands identification  and the primer design  [All the experiments were performed according to protocols described previously . The pri design  .With the informed consent of all patients and approval of the ethics committee, the tumor samples were collected from astrocytoma patients (n = 53) during operation at the Tiantan Neurosurgical Hospital in Beijing. The pathological classification of tumor tissues was carried out and the stage of each astrocytoma patients was determined according to the WHO classification . No sign260 nm readings.Total genomic DNA was extracted from frozen tissue specimens (50 – 100 mg) according to a standard protocol with some modifications ,27. Froz4OAC and 300 μl ethanol. After washing with 70% ethanol, the gently dried DNA pellet was dissolved with 30 μl TE at 65°C for 10 min. The DNA sample was finally stored at -20°C until further use. PCR reaction was carried out in a volume of 15 μl with 50 ng or less template DNA with FastStart Taq polymerase (Roche, Germany) as follows. After an initial heat denaturing step 4 minutes treatment at 94°C, 30 cycles of 92°C for 15 sec, varying temperatures with primer pairs , and only the verified targets were included for the study of the astrocytoma tissues.To optimize the MSP procedure, the M. Sss I treated DNA was used as the methylated control template. In detail, the DNA from a normal liver tissue of the healthy liver donor ,24 was b, where both Pearsong's Chi-square test with Upton's adjustment and Fisher exact test  were used to examine the tissue samples with the low expected values. The relative frequency with a 95% confidence interval (P < 0.05) for a binomial distribution was calculated for the whole as well as each subtype of astrocytoma patients.The methylation data were dichotomized as 1 for the co-existence of the methylated and unmethylated alleles, 2 for methylated allele only and 0 for the unmethylated for both alleles to facilitate statistical analysis using contingency tables. The methylation profiles of each individual gene (in percentage) classified by the genders and grading of the patients were presented both in table and in plot. The statistic analyses for the association between the methylation profile of the gene and each of the clinical-pathological parameters were carried out with the statistics package The concordant methylation behavior of the genes was established by comparing frequency of co-occurrence of 2 to 3 target subsets with a mathematic method, namely Discovery of Association Rules , which i2 atmosphere. When cell culture reached 50% confluence, they were treated with 5-Aza-2'-deoxycytidine (Sigma A3656) at the final concentration 10 and 20 nM, respectively for 3 days. The primer pairs for the RT-PCR , and cDNA was obtained using the Supertranscript plus reverse transcriptase with the oligo-dT as primers. PCR with single pair of the target primers run for 15 cycles, followed by another 15 cycle PCR reactions in the presence of the beta-actin primers (Table U251 cells (an established glioma cell line) were cultured in DEME plus 10% new born calf serum at 37°C in a 5% COTo establish the methylation profile of thirty four genes during the process of astrocytoma development, we recruited 53 astrocytoma patients  for this study. 14 cases were pathologically classified at the Grade I pilocytic astrocytoma , 15 cases at the Grade II diffuse astrocytoma , 12 cases at the Grade III anaplastic astrocytoma , and 12 cases (including 4 recurrent cases) at the Grade IV glioblastoma   as template would be an ideal positive control for the absence of methylated targets in tumor tissue samples. By taking extra caution, we carried out MSP of all the targets with the M. Sss I treated normal liver DNA as positive control templates, except for the MAGEA1 gene was unmethylated in the normal liver tissue. While only the PCR reaction designated to the unmethylated template gave rise to the detectable bands with the parental DNA, the PCR bands were evident in both reactions with the M. Sss I treated DNA (The methylation-specific PCR (MSP) is widely used for methylation profiling of the genes in human cancers for both its easiness and sensitivity. However, the necessary steps have to be taken to eliminate both false positive and negative results. Comparing the MSP-data with the non-PCR data by Southern analysis of the methylation sensitive restriction enzyme is a valuable choice, as our previous work where the hypomethylated status of both analysis . Alternaated DNA . TherefoARF, p15INK4b, p16INK4a RASSF1A, RB1 APC, BRCA1, CDH1, DAPK1, hMLH1, p14and VHL  [OCT6) [MYOD1) [RB1/p16INK4a pathway (ARF, p15INK4b, p16INK4ap14, and RB1) [p27KIP1 [p57KIP2) [p53 analogue:(p73) [RASSF1A, and RASSF1C [VHL [APC [PTEN [DBCCR1) [WT1) [MAGEA1) [CAV) [CSPG2) [AR) [CDH1 [CDH13) [IRF7), and a serine/threonine kinase 1 (Peutz-Jeghers syndrome) gene (LKB1) [MGMT) [MT1A) [hMLH1 [BRCA1 [APAF1 [DAPK1 [SURVIVIN [ABL) [Eleven of the thirty four target genes were previously studied either in astrocytoma or other types of tumors. The published PCR conditions for these genes:  and VHL  were adoordingly ,24. Somely [EPO) , a ubiqu) [OCT6) , and the [MYOD1) . The majand RB1) , and two[p27KIP1  and p57Kp57KIP2) . Other gue:(p73) ,35, two  RASSF1C ), VHL [3F1C [VHL , APC [38[DBCCR1) , and the1) [WT1) . We incl[MAGEA1) , caveoli1) [CAV) , chondro [CSPG2) , androgeG2) [AR) , and cadR) [CDH1  and CDH1 [CDH13) . Three g [CDH13) , the int) [MT1A)  which pl) [hMLH1 , and BRC1 [BRCA1 , while f1 [APAF1 , DAPK1 [SURVIVIN ). FinallIN [ABL)  (AdditioABL(1), APAF1(2), APC(3), BRCA1(5), CAV(6), CDH13(8), DAPK1(11), hMLH1(14), LKB1(16), p14ARF(22), p15INK4b(23), p27KIP1(25), p57KIP2(26), PTEN (28), RASSF1C(30), RB1(31), SURVIVIN(32), and VHL(33) genes , hMLH11, LKB1163), BRCA1, CAV(6),3, BRCA1,6), CDH13, DAPK1(13) genes .Lack of hypermethylation of the RB1 gene in our observation was inconsistent with a recent report that the hypermethylated RB1 gene was detected in 19% of astrocytoma patients  . Since tPTEN gene contributed to the pathogenesis of astrocytoma [PTEN inactivation that occurs frequently in astrocytoma. This explanation might also be applicable to the no change type of methylation behavior for both the tumor associated genes (ABL(1), APAF1(2), APC(3), BRCA1(5), CAV(6), CDH13 (8), DAPK1(11), hMLH1(14), LKB1(16), p14ARF(22), p15INK4b(23), p27KIP1(25), p57KIP2(26), PTEN (28), RASSF1C(30), RB1(31), SURVIVIN(32), and VHL(33) genes) and the "cancer unrelated" genes (EPO (14)) (The genetic defects affecting the rocytoma . Lack of), hMLH11, LKB1163), BRCA1, CAV(6),3, BRCA1,), CDH13 , DAPK1(1PO (14)) .CDH1 (7), CSPG2(9), cyclin a1(10), DBCCR1(12), IRF7(15), MGMT(18), MT1A(19), MYOD1(20), OCT6(21), p16INK4a (24), p73(27), RASSF1A (39) and WT1(34)) were unmethylated in all three normal controls. In contrast, these genes were hypermethylated to various extents in the astrocytoma samples. The following six genes were marginally hypermethylated: p16INK4a, EPO, DBCCR1 and MYOD1 genes were hypermethylated in 1.9% (1/53) of astrocytoma tissues, while both CDH13 and cyclin a1 genes were hypermethylated in 5.7% (3/53) of astrocytoma cases. No significant changes of these six genes shown in here acted against the notion that DNA methylation related mechanisms underline potential inactivation of this set of genes in the pathogenesis of astrocytoma. The infrequent hypermethylation of the p16INK4a gene in astrocytoma was a total surprise, as it was frequently reported hypermethylated in various human tumors tested, including in HCC where we have previously found that the p16INK4a, MYOD1, CDH13 and cyclin a1 genes were frequently methylated [p16INK4a gene in astrocytoma was unlikely incorrect, which is consistent with a recent report that inactivation of the p16INK4a gene in 48% of astrocytoma cases was genetic [As shown in 15), MGMT, MT1A(19p16INK4a , p73(27) genetic .OCT6 gene was hypermethylated in 30.2% of the astrocytoma cases (16/53). Despite of the association of the OCT6 methylation with the aging process reported previously, we found no significant correlation/association of the OCT6 methylation to any clinical-pathological features, including age, gender and clinical grading of the patients. The significance of such a prevalent occurrence of the hypermethylated OCT6 gene remains to be determined. The RASSF1A (hypermethylated in 37/53 cases, 69.8%) is a variant of the recently identified tumor suppressor, the RASSF1 gene that acts at downstream of the Ras mediated apoptotic pathway and is capable of binding to Ras in a GTP dependent manner [RASSF1A gene has a more extended 5' part and its promoter CpG island displays a tumor specific hypermethylated profile in a variety of tumors, HCC in particular. Furthermore, lack of the RASSF1A expression in nineteen established tumor cell lines correlates with the hypermethylated state of its promoter CpG island [RASSF1C gene has its own promoter CpG island, but is not methylated in any tumors. The methylation behavior of these two genes was very similar to our previous observation in hepatocellular carcinoma, where 22/29 cases (79%) had the fully methylated 1A along with the unmethylated 1C variants [RASSF1A promoter-CpG island was methylated in 69.8% (37/53) of astrocytoma tissues, while the C variant was not methylated in any astrocytoma tissues. The hypermethylated state of the RASSF1A promoter CpG island was not correlated with gender, age and clinical grading. Consistent with the hypermethylated status of the RASSF1A gene in U251 cells, no expression at the mRNA level was detected. Partial demethylation of its promoter by the treatment with 5-Aza-2'-deoxycytidine indeed resulted in its transcription  of astrocytoma cases. The t manner . The RASG island . The RASvariants . As show3 of astrp73 gene encodes a homologue to TP53, and loss of its heterozygosity has been observed in up to 90% of oligodendrogliomas and in 10–25% of diffuse astrocytoma [p73 gene was prevalently methylated (25/53, 47.2%) with no significant association with any clinical-pathological parameters, such as gender and the WHO grading. The occurrence of the hypermethylated p73 gene was more prevalent in our results than a recent report which detected the hypermethylated p73 gene in 18% (5 /28) of the WHO grade IV but not in grade III astrocytoma [p73 transcription  of cases, implying its possible involvement in the formation of astrocytoma.Both genetic defects and epigenetic abnormalities of the 's tumor . In thisIRF7) gene was analyzed. The analogue (IRF1) of IRF7 has been implicated in the IFN gamma mediated apoptosis with a profound effect on the chemo-sensitivity of tumor cells [2 = 6.632, P = 0.014). Although the gender difference remains to be understood, such a strong male association with IRF7 hypermethylation may have prognostic value.Tumor resistance to the cytotoxic chemotherapies may result from the disrupted apoptosis programs and remains a major obstacle in cancer treatment. In this study, the interferon regulatory factor 7 , and its transcription could be reactivated by demethylation with 5-Aza-2'-deoxycytidine in U251 cells  is inducible by a number of adversary agents such as heavy metals and oxidative stress. Both basal and inducible expression of this gene has been impaired in various tumor cell lines and attributed to the hypermethylated state of this gene [MT1A gene was hypermethylated in 30% (16/53) of cases, with no significant gender and grading difference. The functional and pathological implications of the MT1A hypermethylation in astrocytoma remain to be established.O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic stress. Silencing of the  and p53  and is a and p53 . We founGMT gene . Expresshis gene . In thisCDH1) and H-cadherin (CDH13) has been found in parallel with the hypermethylated promoter CpG islands in various cancers [CDH1 gene was hypermethylated in 32.8% (17/53) of astrocytoma tissues, while the CDH13 gene was not methylated in all the astrocytoma tissues examined . However, its unmethylated status in all the non-astrocytoma tissues acts odd with the notion that its demethylation is associated with pathogenesis in human ovarian cancer reported previously [Although the hypermethylation mediated gene silencing of the tumor suppressor genes is at the focal point of the epigenetic studies, demethylated status of the promoter CpG islands has been linked to the tumor associated activation of the normally silenced genes -23. Ther1 in HCC  and for MAGEA1 gene [MAGEA1 gene was fully hypermethylated in all four cases of the normal liver tissues but significantly demethylated in HCC tissues (21/28, 75%). It was found fully hypermethylated in all the three control tissues and in 74.5% (40/53) of the astrocytoma tissues and partially hypermethylated (13/53, 25.5%) in the other astrocytoma tissues. The occurrence of the MAGEA1 demethylation in HCC differed significantly from astrocytoma (75% verse 25.5%, P < 0.001). As it was fully methylated in the normal tissue, the partial hypermethylation  would imply that the event resulting in the loss of the hypermethylation state of the MAGEA1 gene indeed occurred in astrocytoma and should be scored positive for the changes in the methylation pattern in this study. The same principle has been applied for the opposite changes from the unmethylated pattern in the normal control to the partial or full hypermethylated status of all the other genes in astrocytoma tissues. It was also found that the partial demethylated status of the MAGEA1 gene in U251 cells induced by 5-Aza-2'-deoxycytidine occurred co-currently with activation of its transcription  with the clinical pathological parameters (age, grading and gender) were assessed. The methylation profiles of the AR and IRF7 genes were found gender-oriented.By statistic analysis with both Pearson Chi-Square and Fisher's Exact tests, associations of the DNA methylation profiles of the targets displaying no less than 24.5% changes . It may simply be gender associated and do not have any significant relevance to carcinogenesis of astrocytoma. It was also noticed that hypermethylation of the IRF7 gene displayed an opposite gender inclination, detected in 11% of the female patients (3/27), and 42% of male patients (11/26, χ2 = ?6.632, P = 0.014). Despite of the difficulty to offer a mechanistic interpretation, the potential prognostic value of such a gender-associated phenomenon might be worthwhile exploring in future.The  cancers . PhysiolThe hypermethylated status of the promoter CpG island has been linked to gene transcription silencing in a number of biological settings. The effect of the astrocytoma associated changes in the methylated state of the promoter CpG islands detected in this study on gene expression was assessed in U251 astrocytoma cells treated with the a demethylating agent, 5-Aza-2'-deoxycytidine. We used MSP to establish the methylation status of the promoter CpG island of all the genes with the astrocytoma associated methylation changes ,7,8,9 inCDH13, MAGEA1 and p73 genes were heterozygously methylated, both MGMT and RASSF1A genes were fully hypermethylated in U251 cells. The CDH13 gene was found expressed, while the rest transcriptionally inert as measured by the RT-PCR. Although both methylated and unmethylated alleles for p73 and MGMT genes were evident in U251 cells, no expression was detected, indicating that the unmethylated allele may remain silent by the other mechanisms, including the genetic defects at critical control region. By the 5-Aza-2'-deoxycytidine treatment, both demethylation of the promoter CpG island and activation of transcription of these five genes were achieved  or elevated (the CDH13 gene).As shown in panel 1 of Fig. INK4a, and RASSF1A genes, has been well established in human tumors [The DNA methylation mediated epigenetic changes also display the tumor type specific patterns, which seem to reflect the differentiation and maturation histories of the cell lineages as well as the aging process during which both global hypo- and local hyper-methylation occur. Hypermethylation of the promoter CpG islands in accord with the transcriptional silencing of the tumor suppressor genes, such as the p16n tumors . Howevern tumors , based uRASSF1A and p73 genes, hypermethylation of which was found in 20 (37.7%). Three genes that changed together were the former two plus CDH1 or OCT6, hypermethylation of which occurred in 20.8% cases (11/53) (Column 2, a, Four of 53 cases (7.5%) maintained the same methylation profile as the normal control. The rest 49 cases (92.5%) suffered from the methylation changes as much as no less than one target, an occurrence was significantly lower than in HCC, where all the cases displayed methylation changes affecting no less than three targets in the studies involved with twenty or twenty four targets ,24, indiAR is associated closely with the female gender of the astrocytoma patients and devoid of any association with the formation of astrocytoma, it was taken out from this analysis. Hypermethylation of the RASSF1A gene occurred in 21 female cases (77.8%, 21/27). Both RASSF1A and WT1 were hypermethylated in 13 (13/27, 48.1%); and the former two plus the hypermethylated p73 or CDH1 or OCT6 were found in 9 female cases (9/27, 33.3%), respectively (Column 2, b, RASSF1A (16/26, 61.5%); the RASSF1A and IRF7 (10/26, 38.5%); and the former two plus the p73 or MGMT or MT1A (5/26, 19.2%), respectively (c, RASSF1A (10/14, 71.4%), RASSF1A plus p73 (6/14, 42.9%), and the former two plus either WT1 or IRF7 or MAGEA1 as well as RASSF1A plus CDH1 and WT1 (3/14, 21.4%). For Grade II astrocytoma, the corresponding sets consisted of the RASSF1A (12/15, 80%), the RASSF1A and MGMT or IRF7 (5/15, 33.3%), and the RASSF1A and MGMT plus p73 or OCT6, or MT1A, or WT1 as well as the RASSF1A and IRF7 and MT1A (3/15, 20%), respectively. For Grading III astrocytoma, those subsets were composed of the RASSF1A (8/12, 66.7%), the RASSF1A and CDH1 (5/12, 41.7%), and the formal two plus MGMT (4/12, 33.3%), respectively. For Grading IV astrocytoma, the comparative subsets contained the RASSF1A or p73 (7/12, 58.3%), the RASSF1A and p73 (6/12, 50%), and the former two plus MGMT or OCT6 (4/12, 33.3%), respectively. ; cyclin a1: cyclin A1; DAPK1: death-associated protein kinase 1; DBCCR1: deleted in bladder cancer chromosome region candidate 1; EPO: erythropoietin; hMLH1: mutL homolog 1, colon cancer, nonpolyposis type 2; IRF7: interferon regulatory factor 7; LKB1: serine/threonine kinase 11 (Peutz-Jeghers syndrome); MAGEA1: melanoma antigen, family A, 1 (directs expression of antigen MZ2-E); MGMT: O-6-methylguanine-DNA methyltransferase; MT1A: metallothionein 1A ; MYOD1: myogenic factor 3; OCT6: POU domain, class 3, transcription factor 1; p14ARF: the alternative reading frame of the cyclin-dependent kinase inhibitor 4a; p15INK4b: cyclin-dependent kinase inhibitor 4b; p16INK4a: cyclin-dependent kinase inhibitor 4a; p27KIP1: cyclin-dependent kinase inhibitor 1B (p27, KIP1); p57KIP2: cyclin-dependent kinase inhibitor 1C (p57, KIP2); p73: tumor protein p73; PTEN: phosphatase and tensin homolog; RASSF1A: ras association  domain family 1 protein isoform 1a; RASSF1C: ras association  domain family 1 protein isoform 1c; RB1: retinoblastoma 1; VHL: von Hippel-Lindau syndrome; WT1: Wilms tumor 1.HCC: Hepatocellular carcinoma; PCR: polymerase chain reaction; MSP: methylation specific PCR; None declared.JY, HYZ, JG, executing the experiments;SL and JHL, providing the patient samples;WL and YFW, carrying out the mathematic analyses of the dataJDZ: designing and organizing experiments as well as completing manuscript.The pre-publication history for this paper can be accessed here:The target promoter CpG islands and the primer pairs for methylation specific PCR. This file contains his study.Click here for filein vitro methylated genomic DNA by M. Sss I methyl transferase. The Eco RI restricted genomic DNA from the liver tissue of a healthy donor was in vitro methylated overnight with M. Sss I methyl transferase and subjected to the MSP analysis, followed by electrophoresis in a 1.3% agarose gel. *, the DNA size markers, NL, the untreated sample, U and M, MSP with the pair of primers specific to the unmethylated and methylated targets, respectively. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1; 12, DBCCR1; 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MGMT; 18, MT1A; 19, MYOD1; 20, OCT6; 21, p14ARF; 22, p15INK4b; 23, p16INK4a; 24, p27 KIP1; 25, p57KIP2; 26, p73; 27, PTEN; 28, RASSF1A; 29, RASSF1C; 30, RB1; 31, SURVIVIN; 32, VHL and 33, WT1.Methylation profiles of thirty three genes on the Click here for fileMethylation profiles of thirty four genes in astrocytoma (part I). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 1, ABL; 2, APAF1; 3, APC; 4, AR; 5, BRCA1; 6, CAV; 7, CDH1; 8, CDH13; 9, CSPG2; 10, cyclin a1; 11, DAPK1 and 12, DBCCR1.Click here for fileARF>.Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part II). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 13, EPO; 14, hMLH1; 15, IRF7; 16, LKB1; 17, MAGEA1; 18, MGMT; 19, MT1A; 20, MYOD1; 21, OCT6 and 22, p14Click here for fileINK4b; 24, p16INK4a; 25, p27 KIP1; 26, p57KIP2; 27, p73; 28, PTEN; 29, RASSF1A; 30, RASSF1C; 31, RB1; 32, SURVIVIN; 33, VHL and 34, WT1.Methylation profiles of the promoter CpG islands of thirty four genes in astrocytoma (part III). Both electrophoretic patterns of the representative PCR products of each of thirty four targets (indicated respectively, at the top of figures) and the sequencing verification of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. *, size markers, the bands of 250 bp and 100 bp were shown. U, the unmethylated; M, the hypermethylated. Panels: 23, p15Click here for fileThe summary of the astrocytoma cases displaying no or changes in the methylation profiles (part I). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets (except for the MAGEA1 gene) among the total cases was scored for positive changes in astrocytoma. The MAGEA1 was fully methylated (3/3, 100%) in the control, and become partially demethylated in some astrocytoma, therefore, demethylation of the MAGEA1 in astrocytoma was scored as positive changes. Sub-tables: a, the female patient group, b, the male patient group, and c, the control.Click here for fileA1, where the heterozygously hypermethylated) among the total cases was presented in the plot.The summary of the astrocytoma cases displaying no or changes in the methylation profiles (part II). The frequency (%) of the astrocytoma displaying no or the changes in the methylation profile of each target from the normal control were counted and presented in table as well as plotted in the figure below. Sub-tables d-h, the WHO grading I to IV, respectively; The filled, shading and empty boxes indicate the cases where only hypermethylated allele, both hypermethylated and unmethylated alleles and only unmethylated alleles were detected, respectively. The frequency (%) of the hypermethylated targets .Click here for fileThe summary of changes in the methylation pattern in subsets. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in one to nine genes are presented in % and (case number) in the top half of table, which was also plotted. Both occurrence (case number) and frequency (%) for the subsets having no change in methylation and changes in, at least, one to nine genes are presented in % and (case number) in the bottom half of table.Click here for fileThe summary of the concordant methylation behavior of the hypermethylated targets in astrocytoma. The co-occurrence  and frequency (%) of a panel subsets consisting of one to three targets were treated with method "Discovery Association Rules" and presented. Sub-tables: a, the total, b, the female, c, the male, and d-g, the grade I to IV, respectively. Column 1 is the number of target in each subset. Column 2 is the co-occurrence  (frequency in %). Column 3 is the occurrence of any single target in each subsets, presented in case number (frequency %). The column 4 is the gene(s) in subset. N.B., In view of the strong female inclination of the AR methylation and lacking of any association with astrocytoma, AR has been taken off from this analyses.Click here for file


We report on a woman with previously unknown left atrial myxoma, who underwent percutaneous coronary intervention. 45 months after the initial coronary angiography, echocardiography demonstrated a large atrial myxoma, which was not seen echocardiographically before. The retrospective analysis of the pre-intervention coronary angiography revealed atypical vessels in the atrial septum, which are interpreted as early signs of myxoma. Primary cardiac tumors are rare disorders, with an incidence of 0.02% at autopsy. Three quarters of the primary tumors of the heart are benign, half of which are myxomas .As noninvasive cardiac imaging becomes widely available, with increasing resolution provided by echocardiography, computed tomography and magnetic resonance imaging, cardiac tumors are being diagnosed more often. Angiography, apart from its preoperative role to rule out concomitant coronary artery disease, is rarely needed for the diagnostic work-up of cardiac tumors.This report describes the delayed presentation of a left atrial myxoma which was not depicted in an initial coronary angiography performed 51 months earlier in a woman with chest pain.A 62-year-old woman with known metabolic syndrome was referred to our clinic to exclude coronary artery disease invasively. She has been experiencing chest pain for four months which has not increased in frequency or duration since it started. She denied pain at rest, nocturnal pain, difficulty breathing, or palpitations. An echocardiographic stress examination revealed significant ischemia with anteroseptal hypokinesia. Cardiac chambers were morphologically normal. The "baseline" echocardiography and stress echo was unsuspicious of a left atrial myxoma Fig. .Coronary angiography revealed coronary artery disease with a stenosis of the proximal left anterior descending coronary artery (LAD) near the left main stem. A stent was implanted in the proximal LAD with no residual stenosis. The ventricle was morphologically normal.A coronary angiography performed 13 months after stent implantation showed no re-stenosis and a normal left ventricle. No echocardiogram was performed at this time 32 months after the control coronary angiography, the patient was readmitted because of increasing dyspnoea and palpitations. A transthoracic echocardiography disclosed a big (70 × 30 mm) mass in the left atrium attached to the interatrial septum. The tumor prolapsed into the left ventricle obstructing the mitral valve orifice  and the circumflex coronary artery (RCX). Fig. Surgery was promptly performed and the tumor was successfully excised. Histology confirmed the diagnosis of a cardiac myxoma.A retrospective analysis of the initial coronary angiographies (baseline, 13 and 45 months) disclosed the atypical vessels in a small area of interatrial septum (Fig. Myxomas are benign but potentially dangerous with disturbance of rhythm, peripheral embolization or mechanical valvular obstruction, of the atrial or ventricular cavity ,3.The site, mobility, and size of the myxoma determine the clinical course. Some authors found no correlation between the size of the tumor and the clinical picture , others Myxomas presenting with systemic embolism or intracavitary obstruction can be easily detected non-invasively. The early depiction of small intracardiac tumors by means of angiography relies on the detection of atypical vessels supplied by branches of the left or right coronary artery. Our case demonstrates that this early angiographic sign is difficult to find. However, vascular malformations are not pathognomonic for myxomas  and stroIt has been reported in the literature that myxomas can be induced by radiation ,13. BetwIn the present case, a retrospective analysis of the patient angiographies disclosed the atypical vessels which were initially overseen. These vessels could have probably been interpreted as an early sign of myxoma.HPD and FK have written the manuscript and have equally contributed to this publication. HPD, VG and WR have performed the coronary angiographies. WK has performed cardiac surgery. HPD, ACB, FK and GB participated in the design and coordination of the final manuscript. All authors have read and approved the final manuscript.


Several reports indicated that non-thermal electromagnetic radiation such as from mobile phones and base stations may promote cancer. Therefore, it was investigated experimentally, whether 900 MHz electromagnetic field exposure influences lymphoma development in a mouse strain that is genetically predisposed to this disease. The AKR/J mice genome carries the AK-virus, which leads within one year to spontaneous development of thymic lymphoblastic lymphoma.320 unrestrained female mice were sham-exposed or exposed  to GSM like 900 MHz electromagnetic fields for 24 hours per day, 7 days per week, at an average whole body specific absorption rate (SAR) value of 0.4 W/kg. Animals were visually checked daily and were weighed and palpated weekly. Starting with an age of 6 months, blood samples were taken monthly from the tail. Animals with signs of disease or with an age of about 46 weeks were sacrificed and a gross necropsy was performed.Electromagnetic field exposure had a significant effect on body weight gain, with higher values in exposed than in sham-exposed animals. However, survival rate and lymphoma incidence did not differ between exposed and sham-exposed mice.These data do not support the hypothesis that exposure to 900 MHz electromagnetic fields is a significant risk factor for developing lymphoma in a genetically predisposed species, even at a relatively high exposure level. The use of mobile phones is increasing worldwide, although electromagnetic fields emitted by mobile phones and base stations are a source of great concern. However, so far it is unclear, if non-thermal exposure has a direct influence on public health. French et al.  developePim1 transgenic mice [It was suggested that non-thermal exposition to high-frequency electromagnetic fields may rather have a tumor promoter than an initiator effect , since Dnic mice , which anic mice .The differences between the studies may indicate that various species or strains as well as cancer types differ in their sensitivity to electromagnetic field exposure. The sensitivity to electromagnetic fields may result from an acquired lower resistance against adverse effects or a genetic predisposition . DiffereAKR mice are widely used in cancer research for their high leukaemia incidence (60–100%) . Mice of320 female AKR/J mice were airfreighted from the Jackson Laboratory (Bar Harbor, ME, USA), at an age of 4–5 weeks. After arrival, animals were randomized and housed in groups of 6 or 7 on softwood bedding (Altromin, Lage, Germany) in polycarbonate cages (40 × 25 × 15 cm, W × L × H, Ebeco, Castrop-Rauxel, Germany), enriched with paper. Mice were allowed free access to mice standard food pellets (type 1324, Altromin) and tap-water. Twice a week cages were cleaned and water changed. Temperature was controlled at 21°C ± 2°C. The light was on a 12 hours light-12 hours dark cycle, with light on at 8 am. No sterilization measurements were taken, since AKR/J mice are not especially sensitive for pathogens. However, to prevent a possible transfection from humans to mice or mice to mice, respectively, masks and gloves were worn, which were sterilized between handling of different cages. Animals were inspected daily for signs of moribundity and were weighed (accuracy ± 0.1 g) and palpated weekly. Starting with an age of 6 months, blood samples were taken monthly from the tail. A tattoo in the ear allowed individual identification. The Bremen state commission for animal welfare according to §8a of the German animal welfare legislation approved the experiments (522-27-11/2-0).In order to accomplish whole body exposure of a large number of non-restrained animals, one approach is to design exposure setups based on radial waveguides. Inside the waveguide the animals are kept in cages which are arranged at a constant distance from a radiating antenna in the centre, thus uniform exposure of the cages can be achieved. Another advantage is that the radial waveguide is an electromagnetic shielded system, on that score no costly shielding of any laboratory is necessary. The height of the waveguide is preferably chosen smaller than half a wavelength . TherebyTherefore, special modifications of the fundamental geometry of radial waveguides had to be performed to avoid the propagation of the unwanted higher order modes. In order to produce a well defined field distribution the cage region was excited only by the fundamental TEM-mode in a first step, i.e. for small radii the plate distance was kept below 14 cm and for larger radii the height was increased to the required value of 17 cm Figure . Since iBoth implemented waveguides (one for exposure, one for sham-exposure) of ca. 4 m diameter and 17 cm vertical plate distance were placed within the same room and carried up to 24 cages measuring 425 mm × 265 mm × 160 mm (L × W × H) each of them housing 6–7 mice. The cage area was covered with trapezoidal lids (3 cages per opening) with wire mesh . The total level was 69 dB (lin), and less than 25 dB at frequencies between 8 and 40 kHz. Thus possible disturbing effects of ultrasound were excluded.2 gas when signs of a developing disease became evident or at an age of about 46 weeks, after a last blood sample was taken. A gross necropsy was performed focusing on main tissues of disease involvement (spleen, thymus and lymph nodes) and tumor infiltration (liver, kidney, lung, brain). Tissues were immersion-fixed in Bouin's solution for up to 24 h and subsequently in ethanol (70%), embedded in paraffin and sectioned at 5 μm. Blood smears were stained with Pappenheim's stain and tissue slices using hematoxylin and eosin. When a mouse was found dead in its cage (5 exposed, 7 sham-exposed mice) a necropsy was performed, but no tissues were fixed.Animals were sacrificed by COGroup mean body weights were tested for a possible exposure influence in dependence of time, using multiple regression analysis (InStat 3.05, GraphPad). An unpaired t-test was applied to compare the loss of weight associated with lymphoma development. Survival curves and lymphoma incidence were plotted according to the method of Kaplan and Meier. Differences between curves were compared using the logrank test, with animals censored, which were still alive at the end of the study (Prism 4.01, GraphPad). Two-way ANOVA was used to test for possible changes in differential leucocytes counts with time and exposure.Statistical significance of differences was tested two-sided at the p ≤ 0.05 level. If not indicated otherwise, data are given as means ± standard error of the mean. The exposure code was broken only after completion of the analyses.Chronic exposure to 900 MHz electromagnetic fields had no influence on the absolute body weight of female AKR/J mice; however, its influence on the relative weight change was significant (p < 0.001) Figure . The rapWater consumption was approximately 4 g per day and mouse, and not different between exposed or sham-exposed animals (data not shown). This value is in accordance to the water intake measurements published by the Jackson Laboratory , and obvSimilar survival rates were seen in both groups of AKR/J mice Figure . The firIn female AKR/J mice lymphoma developed rapidly, usually associated with lymphadenopathy. In 28.2% (exposed) and 30.3% (sham-exposed) of all animals, lymphomas were restricted to the thymus, followed by respiratory distress and protrusion of the eyes. 13 animals (8 exposed, 5 sham-exposed) died in their cages without any earlier sign of distress, although autopsy revealed enlarged mediastinal mass compressing the thoracic space. The lung was affected in 10% of the exposed and sham-exposed animals; 8 exposed and 6 sham-exposed animals developed macroscopically visible metastatic tumors in the liver or spleen Figure . Other cWhen the animals reached an age of about half a year, differential counts of leucocytes were performed monthly using blood smears from tail blood. As seen in Figure The present study was designed to test the hypothesis that 900 MHz electromagnetic fields, modulated according to the global system for mobile communication (GSM), increases the risk of lymphoma incidence in female AKR/J mice. According to the results this hypothesis has to be rejected, since compared with sham-exposed females of this strain, a mean exposition of 0.4 W/kg SAR neither influenced the risk to develop lymphoma, nor the malignancy of the disease, only an influence on the growth pattern of the animals was observed. However, the present experiment does not allow any conclusions about tumor onset or the kinetics of tumor development, since for such type of study animals would have to be sacrificed and examined at fixed intervals irrespective of clinical symptoms.Caenorhabditis elegans [Pim1 mice was not changed due to exposure to electromagnetic fields [Exposure to electromagnetic fields increased the growth rate in the nematode  elegans , but dec elegans . In contc fields ,18. Duriad libitum, and various studies described effects of electromagnetic fields on the nervous system in humans or rodents [Food was available  rodents -39. It mMitochondrial heat production is one of the main energy consuming processes in endotherms like mice ,41. The Pim1 transgenic mice, which are known to develop spontaneous lymphoma with a high incidence rate. Lymphoma risk was found to be significantly higher in the exposed Eμ-Pim1 mice than in the controls, mostly pronounced for non-lymphoblastic lymphoma. Humans are presently not known to carry an activated Pim1 gene, but other inherited gene defects are known that predispose carriers to develop cancer [Pim1 mice from the same supplier (Taconic Farms, New York) as Repacholi et al. [A demonstration that long-term exposure to electromagnetic fields derived from mobile phones or base stations increases the incidence of tumors in animals would provide direct evidence that such radiation is carcinogenic. The most positive evidence of an effect of exposure to high frequency electromagnetic fields similar to that used by mobile phones was reported by Repacholi et al. , using Ep cancer . Howeveri et al. , did noti et al. . Becausei et al. .A Japanese study showed that neither 1.5 GHz nor 929.3 MHz electromagnetic field exposure promotes liver carcinogenesis in a medium-term bioassay system, using partially hepatectomised F344 rats . A monopIn the present study non-restrained mice that are genetically predisposed to develop lymphoma with a high incidence were exposed to a "far-field" similar to both Australian studies ,18, withThe present study does not support the hypothesis that chronic exposure to high-frequency electromagnetic fields, similar to those emitted by mobile phones and base stations promote neoplastic development in the hematopoietic system in genetically predisposed mice. However, we cannot rule out that exposure to electromagnetic fields may be risk factors for other neoplastic development.The authors declare that they have no competing interests.AS carried out the study, performed the statistical analyses and drafted most of the manuscript. AL conceived the study, participated in its design and coordination, and drafted some parts of the manuscript. JS, AB and VH developed the technical set up and delivered the dosimetry for the experiment. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: