Intravenous ATP infusions can be safely administered in the home setting: a study in pre-terminal cancer patients

Summary
Introduction
In recent years favorable effects of adenosine 5′-triphosphate (ATP) on nutritional and functional status and survival in cancer patients were reported [1–5]. However, ATP [6–10] and its breakdown product adenosine [11–18], given either as a continuous intravenous infusion or by intravenous bolus administration, were also reported to have side effects, which might hamper administration especially in an outpatient setting.
In cancer patients, ATP was administered as continuous intravenous infusions over several hours, in doses varying from 25 to 100 μg kg−1 min−1. In a Phase I study [4], 14 patients with advanced cancer received 96-h infusions of ATP once per month for a total of at least three infusions, as tolerated by the patient. In the first three patients, ATP was given at a dose of 50 μg kg−1 min−1. If well tolerated, subsequent dose escalations were planned in groups of three patients in increments of 25 μg kg−1 min−1 until the maximally tolerated dose (MTD) was reached. Patients who completed therapy without serious toxicity and later progressed were eligible for retreatment at a higher dose of ATP. Fourteen patients received a total of 43 ATP infusions: 11 patients received 29 infusions at a dose of 50 μg kg−1 min−1, six patients received 11 ATP infusions at a dose of 75 μg kg−1 min−1, and three patients who had tolerated 50 μg kg−1 min−1 very well were reconsented and received one cycle each at a dose of 100 μg kg−1 min−1. In total, 179 adverse events were reported, whether or not these could be ascribed to ATP. The majority of side effects (dyspnea, chest pain, hypotension, gastrointestinal side effects, dizziness, headache, fever and local injection site reaction) were minor (104 adverse events grade 1 (58%) and 54 adverse events grade 2 (30%) based on the US National Cancer Institute Toxicology Criteria. Dose-limiting toxicities of ATP were reported in seven patients, i.e. asymptomatic electrocardiographic evidence of cardiac ischemia (n = 2), grade 3 cardiac toxicity (angina without evidence of infarction), and grade 4 pulmonary toxicity (dyspnea at rest), usually occurring as a characteristic cardiopulmonary syndrome including an initial feeling of chest ‘tightness’ and a sensation of ‘needing to take a deep breath’ (n = 5). Dose-limiting cardiopulmonary toxicity appeared in all three 100 μg kg−1 min−1 ATP infusions, in four (36%) 75 μg kg−1 min−1 infusions, and in five (17%) 50 μg kg−1 min−1 infusions.
In a subsequent uncontrolled phase II study [5], 15 previously untreated patients with stage IIIB/IV non-small-cell lung cancer (NSCLC) received infusions of ATP at doses of 50 (first course) and 65 μg kg−1 min−1 (subsequent courses) for 96 h every 4 weeks. Reported side effects were, in decreasing frequency, chest pain, dyspnea, coughing, anxiety, injection site pain, chest tightness, headache, insomnia, and hot flushes. According to the authors, six patients reported severe adverse events, of which CTC 4 dyspnea occurred in two patients; this led to the cessation of treatment in five patients [5].
In the first randomized clinical trial with ATP [1], 28 patients with stage IIIB/IV NSCLC received a total of 176 intravenous ATP courses, at doses of maximally 75 μg kg−1 min−1 (tailored to the maximum tolerance of each patient) for 30 h every 2–4 weeks. Twenty-nine percent of the infusions were given at rates of 25–40 μg kg−1 min−1, 27% at 45–60 μg kg−1 min−1 and 44% at 65–75 μg kg−1 min−1 [19]. The most frequent side effects were chest discomfort (15%) and the urge to take a deep breath (10%), both of which resolved within minutes after lowering the ATP dose. Electrocardiography (ECG) was performed in patients with chest pain/discomfort during the ATP infusions, but no ECG changes suggestive of myocardial ischemia were detected [19]. In all of the above studies, ATP was administered on an inpatient basis under medical supervision.
Based on the previously reported favorable effects of ATP on quality of life, nutritional status and survival [1, 3], we initiated a new randomized clinical trial to investigate whether these effects of ATP could be confirmed in patients with different types of cancer in the pre-terminal stage of disease (life expectancy 1–6 months). In view of the short life expectancy of these patients, and based on the favorable safety profile of ATP, we decided to administer ATP in the home setting.
The aim of the present study was to investigate the safety of ATP administration at home in pre-terminal cancer patients. We hypothesized that side effects of ATP infusions would occur mainly during the first ATP infusion, especially during assessment of the MTD of ATP. We also hypothesized that the presence of cardiac disorders and/or lung cancer would lead to a lower MTD, and a higher frequency of side effects.
Patients and methods
Study population and design
Patients were recruited through the Departments of Medical Oncology and Pulmonology of five hospitals in different regions in The Netherlands (Maastricht, Eindhoven, Utrecht and Heerlen) and through general practitioners in the region of Maastricht. Eligible were patients with cytologically or histologically confirmed cancer, for whom medical treatment options were restricted to supportive care, who had a life expectancy <6 months, had a World Health Organization (WHO) performance status 1 or 2, and who at the time of inclusion suffered from at least one of the following complaints: fatigue, anorexia, or weight loss >5% in the previous 6 months. Exclusion criteria were: symptomatic angina pectoris, symptomatic heart failure, atrio-ventricular block (checked by standard ECG in all patients before inclusion), life expectancy of less than 4 weeks, concurrent palliative chemotherapy at the time of inclusion, cognitive dysfunction, and the presence of other diseases hampering adequate follow up. The eligibility of patients was evaluated by a medical oncologist or pulmonologist. After baseline measurements, patients were randomly allocated to ATP or control treatment. The present report on the safety of ATP administration is restricted to patients randomized to ATP treatment. The study was approved by the Ethical Committee of University Hospital Maastricht and Maastricht University, and all patients signed written informed consent prior to the study. Details of the trial design have been published elsewhere [20].
Intervention
Adenosine 5′-triphosphate (ATP-Na2) of >98% purity was purchased from Pharma Waldhof GmbH and co. KG (Düsseldorf, Germany). For each infusion, 2.5 g of ATP were dissolved in 500 ml NaCl 0.9%, sterilized by ultrafiltration (Minisart 0.22 μM), and supplied in sterile containers. Patients received weekly intravenous ATP infusions of 8–12 h, over a period of 8 weeks. All ATP infusions were started at a dose of 20 μg kg−1 min−1 and then increased in steps of 10 μg kg−1 min−1 every 10 min, until a maximum dose of 50 μg kg−1 min−1, or the MTD, if this was lower, had been reached. Thereafter, ATP was infused at a continuous rate. If any side effects occurred, the dose was reduced until side effects disappeared. Blood pressure and pulse were registered before starting the infusion, at 30 min after determining the MTD, and just before the infusion was stopped.
Since initiation of ATP infusions under medical supervision in a clinical setting would facilitate the treatment of possible side effects, the first two ATP infusions were given at the day care center of the participating hospitals. Based on evaluation of safety data in the first 22 patients, in view of the mild character of the noted side effects during the first two infusions in these patients, the Ethical Committee granted permission to administer only the first ATP infusion at the day care center and all subsequent infusions at home. The description below applies to the procedures in case of only one hospital infusion.
At the end of the first infusion, the safety and tolerated dose of ATP was evaluated for each individual patient. Subsequent infusions were given at the patients’ home by an experienced and trained nurse of a specialized infusion team, usually embedded in the regional Community Care Organization. The MTD as determined during the first infusion at the day care center was also the maximum dose for this patient for the next infusions at home. The same rule was applied for subsequent home infusions, so that the infusion rate in any subsequent ATP infusion course was never higher than the MTD during the previous ATP infusion. Patients and their partners were instructed extensively regarding the infusion procedures; also, they were instructed to immediately call the involved infusion team in case of any side effects.
Documentation of side effects and adverse events
Side effects of ATP infusions
Side effects of all ATP infusions, adjustments of dosage schedules, and any other events occurring during the infusions were registered by the infusion nurse, either a. during the infusions, or b. on completion of each infusion. In addition, the researcher called all patients after each infusion to ask whether any side effects had occurred. Side effects were scored using the NCI Common Toxicity Criteria [21] on a four-point scale, according to seriousness. In this system, dyspnea is graded as follows: 0, no change; 1, not defined; 2, dyspnea on significant exertion; 3, dyspnea at normal activity; 4, dyspnea at rest. Since most terminal cancer patients are at rest, dyspnea would in these cases automatically be classified as CTC grade 4, which would misrepresent the actual severity of the symptoms and disregard the intention of the NCI Common Toxicity Criteria (i.e. CTC grade 4 = life-threatening). For this reason, we decided to apply the general toxicity grading of the CTC also for dyspnea, i.e. 1, mild; 2, moderate; 3, severe; and 4, life-threatening.
Based on Agteresch et al. [19], we pre-defined 13 side effects: chest discomfort, urge to take a deep breath, nausea, flushing, light-headedness, dyspnea, headache, sweating, mood alteration-anxiety, palpitations, epistaxis, symptoms of cardiac ischemia, injection side reaction or other (to be specified). In case of persistent or severe chest pain, suspect for potential cardiac ischemia, an ECG was made in order to exclude this.
Statistical analysis
Data were entered in a database using Microsoft Access 2000 and checked for errors by means of double data entry by two independent persons. Data were analyzed using SPSS 13.0 for Windows. Changes in blood pressure and heart rate over time were tested for statistical significance by Student’s paired t test. To test our hypothesis that side effects would especially occur during the first infusion, we calculated the average number of side effects per infusion over all infusions. To investigate whether the MTD and frequency of side effects were dependent on the presence of cardiac disorders and/or lung cancer, linear regression models were fitted. As dependent variables, we used mean MTD per patient over all courses, and frequency of side effects per infusion. As independent variables, we included dummy variables for the presence of cardiac disorders (yes/no), and lung cancer (yes/no).To investigate the relation between the seriousness of side effects (CTC grades 2–4 vs. CTC grade 1) and the presence of cardiac disorders and/or lung cancer, Fisher’s exact test was used. P values of less than 0.05 indicated significance.
Results
Study population
Fifty-one patients with different types of pre-terminal cancer were included in the present study. Baseline characteristics of the patients are listed in Table 1. Mean age was 68.1 years (range: 45–87 years) and the majority of patients were male (69%). The most frequent diagnosis was lung cancer (41%), followed by colon cancer (16%). The majority of patients (65%) had WHO performance status 1, and 35% WHO status 2. Twelve patients (24%) suffered from cardiac disorders.
Table 1Baseline characteristics of ATP-treated patients (n = 51) NumberPercent (%)Age (years)68.1 (45–87)aGender Male3569 Female1631WHO performance scoreb 13365 21835Tumor type Lung2141 Colon816 Gastro-intestinal other612 Prostate510 Other1121Presence of cardiac disorders1224aMean (range)bWHO 1: restricted in physically strenuous activity but ambulatory and able to do light work; WHO 2: ambulatory (not more than 50% in bed) and capable of self-care but unable to carry out any work
Out of 51 patients, six patients died during the intervention period because of disease progression (Fig. 1). Three patients did not start because of rapid deterioration of their condition and 18 patients stopped with the ATP infusions. Reasons to stop were deterioration in medical condition (n = 11), fear of side effects (n = 4), or being unsatisfied with the effect of ATP (n = 3).
Fig. 1Flow diagram of the study
Over the study period of 8 weeks, a total of 266 ATP infusions were administered in 48 patients. Fifteen patients (31%) received one to three ATP courses, nine patients (19%) received four to seven courses, and 24 patients (50%) completed all eight ATP infusions. Eleven infusions (4%) of 266 infusions were given at the lowest dose of 20 μg kg−1 min−1, 85 infusions (32%) at a dose of 25–40 μg kg−1 min−1, and 170 (64%) at the highest dose of 45–50 μg kg−1 min−1.
Heart rate and blood pressure
Heart rate decreased from 86 ± 1 (mean ± SEM) at baseline to 83 ± 1 at 30 min after reaching the MTD (p < 0.001), and thereafter remained constant at 83 ± 1 beats/min, as measured at 8–10 h of ATP administration (comparison with baseline: p < 0.001). Systolic blood pressure decreased from 126 ± 1 at baseline to 124 ± 2 at 30 min (p < 0.001), but then increased to 133 ± 2 mm Hg at 8–10 h of ATP infusion (comparison with baseline: p < 0.001). Diastolic blood pressure also initially decreased from 75 ± 1 mmHg to 72 ± 1 mmHg (p < 0.001), but then increased to 77 ± 1 mm Hg at 8–10 h of ATP infusion (comparison with baseline: p = 0.02).
Side effects
Ten patients out of 48 had no side effects during any of the infusions. Out of 266 infusions, 167 infusions (63%) were without any side effects. In 48 infusions (18%) 1 side effect occurred, in 29 infusions (11%) 2 side effects, and in 22 infusions (8%) >2 side effects. In total, 192 side effects were reported (Fig. 2): 138 side effects (72%) were reported to the nurse, of which 96 during the infusions and 42 afterwards; and 54 (28%) to the researcher when specifically asked for. The average number of side effects per infusion was 0.72 (i.e. 192 side effects in 266 infusions).
Fig. 2Reported side effects of ATP according to location (day care centre vs. home), time of reporting side effects (during vs. after the infusion), and person to whom the side effects were reported (nurse vs. researcher)
At the day care center, 63 side effects were reported in 95 infusions (0.66 side effect per infusion). Of these 63 side effects, 56 (89%) were reported to the nurse, of which 55 during the infusions and 1 afterwards; and 7 (11%) to the researcher when specifically asked for. At home, 129 side effects were reported in 171 infusions (0.75 side effect per infusion); of these, 82 (64%) were reported to the nurse, of which 41 during the infusions and 41 afterwards; and 47 (36%) to the researcher when specifically asked for. All side effects were transient and resolved within minutes after lowering the ATP infusion rate.
In a total of 99 infusions, one or more side effects were reported by patients. In the 51 infusions with one or more side effects reported during ATP administration, the infusion was stopped in 13 infusions (25%), lowered in 30 infusions (59%) and not changed in 8 infusions (16%). Of the latter eight infusions, the side effects had already disappeared before lowering the infusion rate in four infusions, whereas in three infusions the side effects had already been present before the start of the ATP infusions; in one infusion, extravasation occurred, and the ATP infusion was temporarily interrupted. In 48 out of the total of 99 infusions with one or more side effects, the side effects were reported by patients to the nurse or researcher only after completion of the infusion, and the infusion rate was therefore not adapted.
The frequency of specific side effects observed during the ATP infusions is shown in Table 2. Dyspnea was the most frequent side effect, appearing in 36 out of 266 infusions (14%), followed by chest discomfort (12%) and the urge to take a deep breath (11%). No symptoms of cardiac ischemia occurred in any of the infusions.
Table 2Side effects during a total of 266 intravenous ATP cycles in 51 patients; CTC-gradingNo side effectsSide effects according to CTC gradeTotal side effects1234CTC grade unspecifiedNumberPercent of inf. (%)Cardiac ischemia2660000000Chest discomfort2342614013212Dyspnea2303510003614Epistaxis2660000000Flushing2588000083Headache252112001145Injection side reaction2615000052Lightheadedness25492010125Mood alteration-anxiety2624000042Nausea251141000156Othera2352820013112Palpitations2651000010Sweating2602300162Urge to take a deep breath23818100002811Total16122414192In some courses more than one side effect was observedaThe side effects classified as ‘Other’ were cold shivering, feelings of coldness, pain in infusion arm, dry eyes, sleepiness, burning sensation of the throat, vomiting, diarrhea, pain neck, general discomfort and unspecified
From the 192 side effects, one side effect (lightheadedness) occurred at the day care center and was scored by the nurse as CTC 4 because of prolonged stay (one night) at the hospital after the first infusion; there were no abnormalities on ECG. Four side effects were scored as CTC 3 (in all cases chest discomfort), and 22 side effects as CTC 2. Two CTC 3 side effects occurred at the day care center in two patients, with no abnormalities on ECG. Of these, one patient decided to stop with the ATP infusions. The other patient wanted to continue; for safety reasons, all subsequent infusions in this patient were given at the day care center. The remaining two CTC 3 side effects occurred at home: one in a patient during the normal procedure of stepwise increasing the infusion rate by the nurse according to protocol, in the other patient during constant ATP infusion after the nurse had left; in the latter case, the nurse was called by telephone by the patient or informal caregiver and immediately arrived. In both cases, the infusions were stopped immediately for that very day. The first patient decided to continue the ATP infusions; in this patient, all four subsequent infusions until the end of the study were given at the day care center at the highest ATP dose (50 μg kg−1 min−1), and no further side effects occurred. The second patient decided to stop the ATP infusions.
The following CTC 2 side effects were reported by patients:
To the nurse during the infusions, by seven patients in seven infusions:
Urge to breathe deeply, headache, sweating and cold shivering in one patient at the day care center (the same patient with the CTC 3 chest discomfort and CTC 4 lightheadedness)Sweating in one patient at the day care center at the end of the infusion, due to flushing the infusion line (violation of the administration protocol)Pain in the infusion arm (one patient) and dyspnea (one patient), both at the day care centerUrge to breathe deeply, headache and sweating in one patient at home, during the period of stepwise increasing the infusion rate by the nurseChest discomfort (once) and lightheadedness (twice) at home in two patients, who called the home nurseOut of these seven patients with CTC 2 side effects, two patients decided to stop the ATP infusions for the rest of the study.To the nurse after completion of the infusion, by three patients (four infusions)To the researcher when specifically asked for, by one patient (five infusions)These nine CTC 2 side effects (eight times urge to breathe deeply, once nausea), were probably not sufficiently serious to call the home nurse. All of these patients continued the ATP infusions.
Timing of side effects
Side effects were not only present during the first infusion but also during subsequent infusions, even though the number of side effects per infusion appeared to be lower during the last three infusions (Fig. 3). At the day care center, of the 55 side effects reported during the infusions, 28 (51%) were reported during MTD assessment and 27 side effects (49%) after MTD assessment had been completed (Fig. 2). At home, 41 side effects were reported during the infusion, 22 side effects (54%) during MTD assessment, and 19 side effects (46%) after MTD assessment had been completed. In the latter 19 cases of side effects, the nurse (and once the general practitioner) was called by the patient and visited the patient to lower or stop the infusion rate.
Fig. 3Average number of side effects per ATP infusion in subsequent infusions
Effect of cardiac or lung disorders on MTD and frequency of side effects
Mean MTD was 48 ± 2 (mean ± SEM) μg kg−1 min−1 in patients without cardiac disorders and/or lung cancer (Table 3). In the presence of cardiac disorders mean MTD was 8 μg kg−1 min−1 lower (p < 0.001) and 4 μg kg−1 min−1 lower in the presence of lung cancer (p = 0.02). The mean number of side effects per infusion was 0.71 ± 0.28 in patients without cardiac disorders and/or lung cancer. In the presence of cardiac disorders, the number of side effects was significantly higher: 2.25 ± 0.49 (p = 0.02). The presence of lung cancer had no significant impact on the frequency of side effects (0.66 ± 0.34). Neither the presence of cardiac disorders, nor the presence of lung cancer had a significant impact on the frequency of specific complaints (chest discomfort, urge to breathe deeply, nausea, lightheadedness, or dyspnea). However, in the presence of cardiac disorders, the number of other side effects was significantly higher (p = 0.006). The proportion of CTC grade 2–4 side effects in the presence of cardiac disorders and/or lung cancer (21 CTC grades 2–4 out of 133 side effects, i.e. 16%) was similar to patients without such disorders (seven CTC grades 2–4 out of 59 side effects, i.e. 12%).
Table 3The independent effect of the presence of cardiac disorders and/or lung cancer on MTD and frequency of side effectsMeanNo cardiac disorders and/or lung cancerCardiac disordersLung cancerMTD48 ± 240 ± 3***44 ± 2*All side effects0.71 ± 0.282.25 ± 0.49*0.66 ± 0.34Chest discomfort0.14 ± 0.050.32 ± 0.090.12 ± 0.06Urge to breathe deeply0.08 ± 0.050.25 ± 0.080.09 ± 0.05Nausea0.06 ± 0.040.18 ± 0.070.03 ± 0.05Lightheadedness0.06 ± 0.040.14 ± 0.080.06 ± 0.05Dyspnea0.12 ± 0.050.35 ± 0.090.17 ± 0.06Other0.09 ± 0.060.42 ± 0.11**0.05 ± 0.07Reference group: patients with no cardiac disorders and/or lung cancer*p < 0.05**p < 0.01***p < 0.001
Continuation of infusions after completion of the study
Of the 24 patients who completed all eight ATP infusions, 12 decided to continue ATP administration (nine patients with lung cancer, one with prostate cancer, one with a malignant melanoma, one with liver cancer). Until the censoring date, these patients had received a total of 116 ATP infusions after completion of the regular 8-week study period (range 1–34 additional infusions). Of these 116 infusions, 11 infusions (10%) reached a dose of ≤20 μg kg−1 min−1, 72 infusions (62%) were given at a dose of 25–40 μg kg−1 min−1, and 33 (28%) reached a dose of 45–50 μg kg−1 min−1 ATP. A total of 38 side effects were reported over 116 infusions (0.33 side effects per infusion), a number significantly lower (p < 0.001) than in the first 8 infusions in the same 12 patients.
Discussion
The aim of this study was to investigate whether ATP can be safely administered to patients with pre-terminal cancer in the home setting. A total of 266 infusions were administered for 8–12 h. Although statistically significant changes in blood pressure and heart rate were observed, these were minor and clinically irrelevant, corroborating earlier results [19]. In total, 192 side effects were reported during the 266 ATP infusions, i.e. an average of 0.72 side effects per infusion. Most side effects were mild and transient, presumably due to the short plasma half-life of ATP and adenosine (0.6–1.5 s) [22].
We saw more side effects than in a previous study in 28 lung cancer patients by Agteresch et al. [19]. These authors observed 91 side effects during a total of 176 ATP cycles of 30 h (0.52 side effects per infusion), even though these infusions were administered for a longer period of time (30 h vs 8–10 h in our study). One possible explanation of this discrepancy may be the different procedures of data collection in these two studies. For safety reasons, the safety procedure adopted in the present study was that the researcher contacted the patients after each home infusion to ask for side effects, specifically naming a number of the most common potential side effects (based on side effects listed by Agteresch et al. [19]. Our results clearly demonstrate that this procedure lead to a higher frequency of reported side effects in our study: out of a total number of 192 side effects, 54 side effects (28%) were reported only afterwards to the researcher when specifically asked for. If these side effects (i.e. side effects which had not spontaneously been reported to the nurse during or after the infusion) are excluded, the frequency of the remaining side effects per infusion in the present study (138/266; 0.52) is remarkably similar to the report by Agteresch et al. [19] (91/176; 0.52), in which trained research nurses in a dedicated clinical research unit registered the side effects.
In both our study and the studies by Haskell et al. [4, 5] and Agteresch et al. [19], chest discomfort/pain, dyspnea and the urge to take a deep breath were frequent side effects.
Results showed that in 43 out of 51 infusions (84%) with side effects reported during ATP administration, the infusion rate was lowered or stopped according to protocol. Reasons for not lowering the infusion rate in eight infusions were the fact that in four infusions complaints disappeared automatically; in three infusions the reported complaints were already present before the start of the infusion; and in one infusion extravasation occurred with a temporary interruption of the ATP infusion.
In 48 infusions with side effects, side effects had only been reported after completion of the concerned ATP infusion and the infusion rate was therefore not adapted. One potential explanation for this finding may be that patients were eager to receive ATP at the highest possible dose, as some patients expressed their worry that the potential effectiveness of ATP could fade in case of lowering the dose (similar to chemotherapy), despite our explicit explanation to patients prior to the study that there is currently no evidence that the efficacy of ATP would be better at higher ATP doses. Another possible explanation is that patients in our study population were used to experiencing far more serious complaints caused by progression of the disease and/or previous antitumor treatment.
One CTC 4 side effect, four CTC 3 side effects, and 22 CTC score 2 were observed. The CTC 4 side effect (see “Results”), and two CTC 3 side effects (chest discomfort) occurred at the day care center, with no abnormalities on ECG. The other two CTC 3 side effects occurred at home, one with the nurse still being present; and in the other case, the nurse immediately arrived.
Side effects were not limited to the period of MTD assessment, nor were they limited to the first ATP infusion. The implication of this finding is that alertness with regard to potential side effects of ATP administration is necessary during all subsequent ATP infusions, and not just during the first infusion or MTD assessment.
The lower MTD found in the presence of cardiac disorders or lung cancer indicates that patients with these disorders tolerate lower doses of ATP without side effects than patients without these disorders. Moreover, even at the lower MTD, side effects tend to be more frequent in the presence of cardiac disorders. Because all patients with presence of COPD also had cardiac disorders or lung cancer, this subgroup was not analyzed separately.
Based on our findings, we conclude that ATP administration at home is safe. First, the majority of infusions (63%) are without side effects, and second, our results confirm a previous study in lung cancer patients [19] showing that side effects that do appear are mild and transient, resolving within minutes after lowering the ATP infusion rate. Nevertheless, it remains possible that some side effects could be prevented by encouraging patients to report any side effects without delay. This emphasizes the need for careful instruction and facilitating patients reporting and monitoring of side effects when ATP infusions are administered at home. As patients with cardiac disorders and lung cancer appear to have a lower MTD and more side effects, these patients should be monitored with special care.

atp
pre-terminal cancer
side effects
home-infusion


Prophylactic Mastectomy in BRCA1/2 Mutation Carriers and Women at Risk of Hereditary Breast Cancer: Long-Term Experiences at the Rotterdam Family Cancer Clinic

Background BRCA1/2 mutation carriers and women from a hereditary breast(/ovarian) cancer family have a highly increased risk of developing breast cancer (BC). Prophylactic mastectomy (PM) results in the greatest BC risk reduction. Long-term data on the efficacy and sequels of PM are scarce.
Women with a germ-line BRCA1 or BRCA2 mutation as well as 50% risk carriers from a hereditary breast(/ovarian) cancer (HB(O)C) family are at increased risk of developing breast and/or ovarian cancer compared with the general population.1–4 Options to reduce this risk are regular surveillance, chemoprevention, or prophylactic surgery. Prophylactic surgery includes prophylactic mastectomy (PM) and/or prophylactic bilateral salpingo-oophorectomy (PBSO). PM implies either a bilateral prophylactic mastectomy (BPM) in high-risk unaffected women as well as in high-risk women with a history of breast cancer (BC) previously treated with breast conserving therapy (BCT), or a contralateral prophylactic mastectomy (CPM) after a unilateral therapeutic mastectomy. Several studies have shown that PM strongly reduces the risk of developing (contralateral) breast cancer, while PBSO reduces the risk of ovarian as well as primary breast cancer.5–10 These strategies therefore have commonly been accepted at this moment as risk-reducing strategies for women being at increased risk of HB(O)C.
PM, however, is a drastic and irreversible intervention, and in case of breast reconstruction (BR), is accompanied by a substantial complication rate.11 Further issues of concern with respect to PM include changes in a woman’s body image and self-esteem, changes of sexual function, and in psychological distress.
At the Rotterdam Family Cancer Clinic, 35–51% of women carrying a BRCA1 or BRCA2 mutation opt for either bilateral or contralateral PM.12,13 For women who are contemplating this intervention, it is imperative to have reliable data on the outcomes of PM in a well-defined cohort to make a good informed decision and to minimize postoperative feelings of deception. At the Rotterdam Family Cancer Clinic there is ample and long-term experience with sufficient numbers of women undergoing PM. We previously reported data concerning the occurrence of breast cancer after BPM in unaffected women with a proven BRCA1/2 mutation,5,14 complications of PM with breast reconstruction,11,15 and psychological aspects of PM in combination with BR.16
In the current analysis, we report on an extended series with longer follow-up of women having undergone a PM at the Rotterdam Family Cancer Clinic because of either a proven BRCA1/2 mutation or a genetic susceptibility (50% risk carriers from a HB(O)C family). Our study sample was large enough to discriminate between unaffected women and women with a history of BC (affected). Special attention is paid to the prevalence of (pre)malignant lesions in prophylactically removed mastectomy specimens. Further, we report on the postoperative complications of PM in combination with breast reconstruction.
PATIENTS AND METHODS
As of the start of the Rotterdam Family Cancer Clinic in 1991, PM and/or PBSO are being discussed as risk-reducing strategies with women at increased risk of hereditary BC and/or ovarian cancer. In early years, PM was discussed with BRCA1/2 mutation carriers as well as with women from a HB(O)C family without a proven mutation (so-called 50% risk carriers), and applied for unaffected as well as affected (with a history of breast cancer) women. Due to the development of more advanced mutation-detection methods enabling the performance of a complete gene mutation screen, there has been a shift in more recent years to discuss the option of PM only with identified mutation carriers. Before 1996, the decision to undergo a PM and/or PBSO was discussed individually by the doctor and the woman in question. As of 1996, women opting for either PM and/or PBSO are additionally discussed in the multidisciplinary Committee on Hereditary Tumors. For this purpose, institutional guidelines concerning the surveillance schedule and indications regarding PM/PBSO have been further elaborated and implemented as of 2000, which were updated as knowledge progressed and more evidence-based data became available.
Before 2000, no additional examinations were performed before PM, irrespective of the individual situation (unaffected/affected; mutation/50% risk carrier). Women were seen biannually for physical examination, while a mammography was performed annually. As of 2000, institutional guidelines from the working party on hereditary tumors recommended to perform clinical breast examination (CBE) and imaging examination within 3 months prior to PM, to minimize the risk of finding unexpected malignant changes at PM. At first, imaging examination consisted of either mammography or magnetic resonance imaging (MRI) scan, while more recently MRI has been preferred. Breast ultrasound (US) and, if necessary, fine-needle aspiration cytology (FNAC) are additionally performed in case lesions are found at CBE or one of the imaging examinations. Further, the guidelines recommend the discussion of the case in the multidisciplinary Committee on Hereditary Tumors and a standard visit with a psychologist. For affected women, the guidelines are extended with dissemination investigations to rule out recurrent or distant breast cancer activity (chest x-ray, liver ultrasound, bone scan, liver functions and determination of Ca15.3/Ca125). Where women with a history of ovarian cancer were previously eligible for PM, at the moment this is not discussed anymore in this setting, because the prognosis is mainly dictated by the ovarian cancer. In the sample, these women were classified as “unaffected,” unless they also had a history of BC.
To evaluate the short-term and long-term medical effects of prophylactic surgery in high-risk women, a combined retrospective and prospective, longitudinal study was activated at our institution, including all genetically susceptible women who had opted for prophylactic surgery (either PM and/or PBSO). Women were informed by oral and written information and were asked for written consent. The protocol was approved by the institutional review board (project EMC-DDHK 98-15).
Surgical Technique
At our institute, the oncological and plastic surgeon perform the PM and BR as a team. During the operation, the patient is under general anesthesia in a half-supine position. A skin-sparing mastectomy is performed through a vertical, peri-areolar incision, which extends from just above the nipple down the submammary fold. The breast tissue, including the superficial fascia (creating thin skin flaps), the axillary tail, the inframammary fold, the nipple-areolar complex, and the fascia of the pectoral muscle are removed. In case of immediate breast reconstruction, either a subpectoral silicone implant is inserted in a pocket created below the pectoral muscles in a one-stage procedure, or autologeous tissue is used. Autologeous reconstruction encompasses a broad range of procedures incorporating the patient’s own tissues to recreate the breast. The transverse rectus abdomis myocutaneous (TRAM) flap and latissimus dorsi flap are two standard myocutaneous flaps used for breast reconstruction. More recent modifications to the traditional techniques led to the use of the deep inferior epigastric perforator (DIEP) flap. Nipple reconstruction is offered after 6 months and consists of three small transposition flaps; the areola is mimicked by tattooing the desired skin color. Breast reconstruction is not always performed in the same operation as the mastectomy; the techniques for these delayed reconstructions, however, are as described previously.
Microscopic Examination of Mastectomy Specimens
As of 1995, a standard procedure has been followed for meticulous microscopic examination of prophylactically removed mastectomy specimens to rule out the presence of “occult” (microscopic) malignant alterations. The protocol prescribes that mastectomy specimens are cut into slices of 0.5–1 cm thickness, whereby each slice is carefully inspected and palpated for abnormalities. Standard, three randomly selected parenchymal tissue samples from each quadrant and a transverse section through the nipple are submitted for histology, in addition to samples of all visible or palpable abnormalities. Further, three samples from each quadrant of the mastectomy specimens are snap frozen for the tissue bank. Radiographic examination of breast tissue specimens is not performed on a routine basis.
Study Design
The current study included all women at increased risk of hereditary BC, according to previously described criteria,17 who underwent prophylactic bilateral or contralateral mastectomy between January 1, 1994 until December 31, 2004. Of our study cohort 310 women (86.6%) underwent PM at our clinic, while 48 women (13.4%) were treated elsewhere, e.g., due to a waiting list at our clinic, or the fact that previous surgery was performed elsewhere. The latter women were only eligible for this analysis if the follow-up after PM took place at our clinic, and a copy of the pathology report was available. In general, DNA testing was performed before the prophylactic surgery, although some women choose for prophylactic surgery without or irrespective of DNA testing. DNA analysis was performed according to standard procedures, as has been previously described.18,19 DNA testing was not an inclusion criterion for participation in the study. Proven noncarriers from a family BRCA mutation were excluded from the study.
Relevant data were extracted from the hospital records. For each woman, including deceased women, the following information was obtained: date of birth, death, and PM, performance (yes/no) and type of breast reconstruction, PBSO, diagnosis of breast and/or ovarian cancer, mutation status, duration of follow-up after PM (end date being either the date of death or the date of last clinic visit in case of loss to follow-up, or the end date of this study, i.e. December 31, 2004), and type and number of complications after breast reconstruction. Regarding the latter, we distinguished between early (within 6 weeks) and late postoperative complications (after 6 weeks). Early complications consisted of infection, necrosis, bleeding, and luxation of the prosthesis. Late complications were divided in surgical complications (such as capsular formation, infection, necrosis, and luxation of the prosthesis), and complaints related to cosmetic outcome (such as poor symmetry and dog ears). Nipple reconstruction is regarded as part of the breast reconstruction and therefore has not been registered as a cosmetic complication. A computerized database (MS-Access) was used to process the data. Data were entered retrospectively as well as prospectively after each clinic visit.
Statistical Analysis
Descriptive statistics (median, range, and frequency) were computed. When appropriate, statistical significance testing between relevant subgroups was performed using the chi-square test for categorical variables and a t-test for continuous variables. A P value of less then .05 was considered statistically significant.
RESULTS
Study Population
In Table 1 the characteristics of the women who underwent a PM are shown. A total of 358 women, with a median follow-up after PM of 4.5 years, fulfilled the study eligibility criteria, consisting of 181 (50.6%) women with a history of breast cancer (affected women), and 177 (49.4%) women without a history of BC (unaffected women). A total of 236 (65.9%) women were BRCA1/2 mutation carriers, while the other 122 women (34.1%) were 50% risk carriers from a HB(O)C family. The unaffected group mainly consisted of BRCA1/2 mutation carriers as compared with 50% risk carriers (82% vs 18%, P < .001), whereas the affected group consisted of an equal number of mutation carriers and 50% risk carriers (91 mutation carriers versus 90 50% risk carriers, P = .94). This difference in distribution was highly significant (P < .001).
TABLE 1.Characteristics of the study populationHISTORY OF BCNo (Unaffected, n = 177)Yes (Affected, n = 181)P valuedBRCAHB(O)CBRCAHB(O)Cn(%)an(%)an(%)an(%)aNo. of women (N)145(82)b32(18)b91(50)c90(50)c<.001e  Death due to cancer0(0)1(3)9(10)7(8)Age at PM (years)  Median36.038.542.047.0<.001  Range22–6528–5525–6526–68  <30 years18(12)3(9)6(7)2(2)<.001  30–39 years74(51)14(44)26(29)22(24)  40–49 years36(25)10(31)38(42)31(34)   ≥50 years17(12)5(16)21(23)35(39)Duration of follow-up (years)4.44.73.94.5Mutation status  BRCA1115(79)–76(84)–  BRCA230(21)–15(16)–PBSO  Yes83(57)3(9)61(67)12(13).13  No62(43)29(91)30(33)78(87)Age at PBSO (years)  Median40.045.043.049.0<.01  Range29–5735–4532–6537–58Timing of PBSO  Before PM18(22)2(67)14(23)6(50).11  At PM35(42)1(33)16(26)5(42)  After PM30(36)0(0)31(51)1(8)Ovarian cancer before PM4(3)0(0)2(2)1(1)Unexpected (p)MF at PM3(2)2(6)4(4)1(1)Cancer after PM  Breast cancer1(1)0(0)0(0)0(0)  Ovarian cancer2(1)0(0)1(1)0(0)BC, breast cancer; HB(O)C, hereditary breast(/ovarian) cancer; PM, prophylactic mastectomy; PBSO, prophylactic bilateral salpingo-oophorectomy; (p)MF, (pre)malignant findings.a Percentage of the number of women in column in question, unless stated otherwise.b Percentage of unaffected women.c Percentage of affected women.d Difference between unaffected and affected BRCA1/2 mutation carriers, unless stated otherwise.e Difference in distribution between unaffected and affected women.
The median age at PM in the unaffected and affected group was significantly different, being 37 and 44 years, respectively (Table 2, P < .001). In the affected group, mutation carriers were significantly younger at PM than 50% risk women (42 vs 47 years, respectively; P = .045). In the unaffected group, we found no significant difference in this respect (36 vs 38.5 years, respectively; P = .102). A history of ovarian cancer was present in four mutation carriers from the unaffected group (2.3%), and in three women (1.7%) in the affected group.
TABLE 2.Comparison between unaffected and affected womenUnaffectedAffectedP valueNo. of women (N)177 (%)181 (%)Age at PM (years)  Median3744<.001  Range22–6525–68  <30 years21 (12) 8 (4)<.001  30–39 years88 (50)48 (27)  40–49 years46 (26)69 (38)  ≥50 years22 (12)56 (31)PBSO  Yes86 (49)73 (40).12  No91 (51)108 (60)Age at PBSO (years)  Median4044<.001  Range29–5732–65PM, prophylactic mastectomy; PBSO, prophylactic bilateral salpingo-oophorectomy.
In Fig. 1, the number of PMs per year, separately for unaffected/affected BRCA mutation carriers, as well as for unaffected/affected 50% risk carriers is shown. In the unaffected group, the annual number of women undergoing PM was always larger in mutation carriers compared with 50% risk carriers, except for the first 2 years (1994/1995). Further, the number of unaffected BRCA mutation carriers undergoing PM widely differed over the years, with two peaks in 1998 and 2001, respectively. In contrast, the number of unaffected 50% risk carriers undergoing PM was quite stable over the years.
FIG. 1.Annual number of PM in unaffected/affected BRCAa/2 mutation carriers and 0% risk carriers from HB(O)C families.
In the affected group, however, the pattern was different, whereby a shift has taken place through the years. In the early years of the study period (1994–1997), mainly women without a proven mutation underwent PM in this group. Between 1997 and 2001, approximately as many mutation carriers as 50% risk carriers underwent a PM. As of 2002, more mutation carriers have undergone a PM, although the number of PMs in 2004 again was not different between mutation and 50% risk carriers.
Prophylactic Bilateral Salpingo-oophorectomy
A considerable part of the BRCA mutation carriers undergoing PM also opted for PBSO, being 57% in the unaffected versus 67% in the affected group, respectively (Table 1, P = .13). In the HB(O)C group, however, only a minority of the women underwent a PBSO, being 15 of 122 50% risk carriers (12%). The median age at PBSO was younger in BRCA mutation carriers compared with the 50% risk carriers, both in the affected and the unaffected group. This difference, however, was not significant (P = .13 and P = .40, respectively). Further, the median age at PBSO was lower in the unaffected compared with the affected group, being 40 versus 44 years, respectively (Table 2, P < .001). In addition, unaffected BRCA mutation carriers underwent PBSO at a younger age compared with mutation carriers with a history of BC (40 vs 43 years, respectively, P < .01, Table 1).
(Pre)malignant Findings at PM
In 10 of the 358 women (2.8%), abnormal findings were unexpectedly found in the mastectomy specimens (table 1). Prior to intended PM there was no suspicion to justify an axillary nodal dissection in combination with the PM procedure. This occurred in five “unaffected” (2.8 %) as well as in five previously affected women (2.8%), and in both mutation carriers (3%) and 50% risk carriers (2.5%).
The characteristics of the unexpected (pre)malignant findings as well as the preoperative screening results are chronologically described in detail in Table 3. In 1995, in one woman both preoperative clinical breast examination (CBE) and mammography were suspicious for a malignancy. However, additional investigation, consisting of ultrasound and cytology, did not reveal a malignancy. Nevertheless, histological examination of the mastectomy specimens revealed an invasive ductal carcinoma (IDC), eventually staged as a pTxN1Mo. The patient died of metastatic breast cancer 4 years after the PM. In another two women, undergoing PM in 1996 and 1997, a lesion was found preoperatively (CBE and mammography, respectively) and classified as probably benign. No malignant abnormalities were seen at subsequent ultrasound examination, which is the reason a FNAC was not performed. However, histological examination of the mastectomy specimens revealed a small ductal carcinoma in situ (DCIS) in both women. In 2002, in one woman preoperative MRI revealed a lesion classified as probably benign. Indeed, no malignant abnormalities were found at additional ultrasound examination. Histological examination of the mastectomy specimens, however, revealed an IDC in the right, and an invasive medullar carcinoma (IMC) in the left breast. Preoperative screening in the remaining six women, performed 1–6 months preceding PM, did not show suspicious abnormalities. Still, another invasive carcinoma, three cases of DCIS, and two cases of lobular carcinoma in situ (LCIS) were found in the mastectomy specimens. All unexpected (pre)malignant findings in the affected women were found in the contralateral breast.
TABLE 3.Characteristics of unexpected (pre)malignant findings in the PM specimensPreoperativeYear of PMGenetic risk groupHistory of BC before PMHistologyGradeTumor size (mm)ER/PR statusCBEMxMRI1995HB(O)CNoIDCNANANASCSCa, b1996HB(O)CNoDCISII<2NAPBnla1996BRCA1NoLCISNANANAnlnl1997HB(O)CYesDCISINANAnlc1997BRCA1YesDCISII<2NAnlPBa1997BRCA1YesIDCII3Negativenlc1998BRCA1YesDCISNA<2NAnlnl2000BRCA1YesLCISNANANAnlnl2002BRCA1NoIDCIII5NegativenlnlPBaIMCIII6Positive2003BRCA2NoDCISII<2NAnlnlPM, prophylactic mastectomy; BC, breast cancer; DCIS, ductal carcinoma in situ; LCIS, lobular carcinoma in situ; IDC, invasive ductal carcinoma; IMC, invasive medullar carcinoma; CBE, clinical breast examination; Mx, mammography; MRI, magnetic resonance imaging; SC, suspicion of cancer; PB, probably benign; nl, normal; NA, not applicable.a Additional investigation normal.b Macroscopic palpable tumor; microscopic no clear border, therefore tumor size and grade not determinable.c Treated previously for BC at another hospital. No information about preoperative imaging available.
Cancer During Follow-up After PM
After PM, no incident breast cancer cases were observed in 50% risk carriers.
In BRCA mutation carriers, ovarian cancer was detected in two women in the unaffected group, and in one woman in the affected group (Table 1).
One BRCA1 mutation carrier from the unaffected group presented in 2001, 3.5 years after PM (no malignant findings at histological examination), with metastatic adenocarcinoma in an axillary lymph node, morphologically and immunohistochemically consistent with breast cancer. Additional dissemination examinations also revealed metastases in bone and liver. Review of the preoperative data (physical examination and mammography) at the presentation of metastatic disease did not show malignant alterations, while meticulous reexamination of all pathology slides and additional investigation of frozen tissue material did not detect a primary breast cancer.
Reconstructive Breast Surgery
In Table 4, the numbers and types of reconstructive breast surgery, as well as the numbers and types of complications after breast reconstruction (BR) are shown. Of the total population (n = 358), 276 women underwent BR in combination with PM, being 60% unaffected and 40% affected women. The presence or absence of a BRCA1/2 mutation did not influence the BR rate (P = .23 for unaffected, and P = .10 for affected women), or the type of BR (P = .25 for unaffected, and P = .68 for affected women). Unaffected women mainly opted for BR (94%), consisting of 163 immediate and three delayed reconstructions, while a considerable part of the affected women did not opt for BR (37%) (P < .001). Further, unaffected women mainly opted for BR by means of (silicon) prosthesis (96%). In contrast, affected women opting for BR (102 immediate, and 8 delayed) more often had a reconstruction by means of autologeous tissue compared with unaffected women, being 13% versus 3%, respectively (P = .004).
TABLE 4.Breast reconstruction (BR) in women undergoing PMHistory of BCNo (Unaffected)Yes (Affected)P valueNumber of women177(%)181(%)Breast reconstruction (BR)  No9(5)68(37)<.001  Yes166(94)110(61)  Unknown a2(1)3(2)Type of BR  Silicon prosthesis159(96)95(86).004  Autologeous tissue6(3)14(13)  Unknown a1(1)1(1)Therapy first BC b  BCT––7(6)  BCT/RT––33(30)  MAST––67(67)  MAST/RT––3(3)Women with complications after BR  No84(51)55(50).92  Yes82(49)55(50)Total number of complications after BR c12788  Early (<6 weeks after BR)42(33)31(35).74  Late (>6 weeks after BR)85(67)57(65)Surgery due to late complication  No38(30)24(27).67  Yes89(70)64(73)Early complicationsSurgery due to early complication  No27(64)17(55).42  Yes15(36)14(45)Type of early complication Infection8(19)14(45).09 Necrosis11(26)4(13) Bleeding20(48)12(39) Prosthesis luxation2(5)0(0) Poor arterial inflow0(0)1(3) Pneumothorax1(2)0(0)Late complicationsSurgery due to late complication  No11(13)7(12).91  Yes74(87)50(88)Type of late complication  Infection4(4)0(0).33  Necrosis1(1)3(5)  Capsular formation31(37)20(35)  Prosthesis luxation2(2)3(5)  Poor cosmetic appearance d31(37)19(34)  Dog ear16(19)12(21)PM, prophylactic mastectomy; BC, breast cancer; BR, breast reconstruction; BCT, breast conserving therapy; BCT/RT, BCT in combination with radiotherapy; MAST, therapeutic mastectomy; MAST/RT, MAST in combination with radiotherapy.a Surgery performed at another hospital, not included in P value calculation.b Data shown for women with a BR after PM.c One woman can have ≥1 complication.dIncluding asymmetry.
In 137 of 276 women opting for BR (49.6%) one or more complications were registered, totaling 215 complications. Surgical reinterventions were performed in 153 cases (124 for late complications).
Concerning the number of complications, this was not different between unaffected and affected women, neither for the moment of the complication (i.e., early or late) (P = .74), the necessity of reintervention due to the complication (P = .67), nor for the type of complication (P = .09 for early complications and P = .33 for late complications).
DISCUSSION
In this study we updated and extended the long-term experiences at the Rotterdam Family Cancer Clinic with prophylactic mastectomy (PM) in proven BRCA1/2 mutation carriers and in 50% risk carriers from a HB(O)C family. We compared the data of PM and breast reconstruction (if performed) in women with (affected) and without a personal history of BC (unaffected women) and further distinguished between women with a BRCA 1 or 2 mutation, and women without a proven mutation. While within the unaffected group, especially BRCA1/2 mutation carriers opted for PM, we observed that within the affected group an equal number of mutation carriers and 50% risk carriers from a HB(O)C family opted for PM. Women carrying a BRCA 1 or 2 mutation are known to have an increased risk of developing contralateral primary BC,20,21 which is even more apparent among women who are younger when diagnosed with a primary breast carcinoma (age <50 years).22,23 For high-risk women without a proven mutation inconsistent results on the risk of developing a contralateral breast cancer (CBC) were reported. Shahedi et al.24 as well as Kirova et al.25 reported an increased risk of developing CBC in non-BRCA1/2 women, while Tilanus et al.26 concluded that the rate of CBC was only slightly and insignificantly increased in non-BRCA1/2 compared with sporadic breast cancer patients. In view of these results, one expects that after a first diagnosis of breast cancer, especially mutation carriers will opt for prophylactic removal of the remaining breast tissue. In our study sample, however, also a considerable part of the women without a proven BRCA1/2 mutation opts for prophylactic mastectomy, especially after a history of breast cancer. It has to be mentioned that, since information on the BRCA1/2 mutation status is not always known in the latter group and genetic testing is missing a number of mutations, it is likely that some of these patients are in fact mutation carriers. Further, the group of women with a history of breast cancer, with and without a gene mutation, may partly consist of women who initially chose for surveillance, and eventually opted for PM after the diagnosis of breast cancer. This is in accordance with an earlier report indicating that women may be more likely to undergo PM after a previous diagnosis of BC.27 This might also partly explain the higher age at the time of PM in the group with a history of BC.
We found that the age at PM was younger in unaffected women, both for mutation carriers as for 50% risk carriers. Further, we found a significant difference between unaffected and affected mutation carriers in the distribution of the numbers of PM over the various age categories, with the highest numbers of PM in the age group of 30–40 for unaffected and 40–50 for affected carriers, respectively. Moreover, the distribution of PM over the various age categories remained completely identical to the age distribution reported in a previous study on PM from our institute,5 indicating consistency over time.
Our data show that, despite preoperative (imaging) examination, the presence of unexpected microscopic (pre)malignant findings in this group of high-risk women is real (3%). Other studies reporting on high-risk and/or pathologic findings in prophylactically removed breast tissues, described percentages varying from 0.1–57%.8,27–29 However, the comparison of frequencies of unexpected (pre)malignant findings between studies is hampered by differences in population selection, preoperative screening methods, pathological examination of the specimens, and definition of what is considered (pre)malignant [e.g., lobular carcinoma in situ (LCIS)]. The percentage of unexpected invasive carcinomas in these studies ranged from 0.1–7.7% (0.8% in our study). Most of these studies, however, did not provide information about the outcome of preoperative physical breast or imaging examination, which at the moment is a standard procedure at our institution. It might be that since the implementation of institutional guidelines concerning preoperative breast examination in 2000, and the introduction of magnetic resonance imaging (MRI), being more sensitive in detecting carcinomas in high-risk women,30,31 as detection tool, the number of unexpected malignant findings in the PM specimens is decreasing.
With a 3% incidence of unexpected microscopic (pre)malignant findings, the potential role of sentinel node biopsy (SN) for all patients undergoing PM has been discussed. However, the majority of the (pre)malignant findings we found in this series, represents DCIS/LCIS; settings for which a sentinel node biopsy is not standardly indicated. Invasive cancer was found in only 0.8% of the patients in this series. Therefore, in our opinion, routine use of SN in all patients undergoing PM is not warranted, which is also supported in the paper by Boughey et al.32
A previous study from our institution, investigating the efficacy of PM in unaffected women with a proven BRCA1/2 mutation, observed no cases of breast cancer after PM.5 The mean follow-up in that study was 3 years. In the current cohort, one BRCA1 mutation carrier presented with metastatic disease 3.5 years after PM (no primary BC found), suggesting the presence of an occult primary tumor that was never found, despite a thorough reexamination of the specimen at the presentation of the metastatic disease. This finding emphasizes the fact that despite thorough examination of the mastectomy specimens, the presence of an occult breast cancer cannot be ruled out completely and indicates that a form of surveillance after PM might be relevant.
The number of reconstructions after risk-reducing mastectomy was lower in the affected group. This may be due to the fact that BR after previous radiotherapy and/or therapeutic mastectomy not always leads to satisfactory cosmetic results.15,33 Some patients abandon, in consultation with and/or at the advice of their (oncological/plastic) surgeon, from BR for this reason. Other women have accepted the mutilation/alteration of body image caused by mastectomy, are reluctant to undergo renewed surgery, and prefer the use of external prosthesis.
During the follow-up period of this study, 49.6% of the women with immediate or delayed BR after PM showed complications. In total 215 complications were registered, leading to surgical reintervention in 153 cases. These findings are consistent with several other reports,34,35 though there are also studies reporting lower,11,15,33 or even higher36 complication rates after (immediate) BR. However, the literature in this area is difficult to compare, in part because not all previous series compare bilateral prophylactic mastectomy in unaffected women with risk-reducing mastectomies in women after a previous therapy for breast cancer. Furthermore, data may not be comparable because of different definitions of complications. Moreover, some studies describe the complication rate as a percentage of the total number of reconstructions,11,15,33 while others, like our study, present the percentage of women with complications.34–36
We found no differences in the numbers of complications after (immediate) breast reconstruction in unaffected women compared with previously affected women in this study. This finding appears to be in contrast with earlier reports (also from our institution) describing the occurrence of more complications after mastectomy followed by (immediate) breast reconstruction in affected women. These studies report negative effects of preoperative radiotherapy on the cosmetic outcome of the reconstruction, in particular the risk of capsular formation would be increased, having negative consequences on the symmetry of the breasts. Further, asymmetry can be expected to occur more often after previous therapeutic mastectomy. Although we have no explanation for our findings, it is possible that the experience of the surgeons at our institution is important. Indeed, where previously BR by means of silicon prosthesis after breast conserving therapy was performed, this is not done anymore.
In summary, we confirmed our previous findings that prophylactic mastectomy strongly reduces the risk of developing breast cancer in both BRCA1/2 mutation carriers and 50% risk carriers. As the frequency of unexpected cancers in this high-risk group remains real, preoperative imaging and careful histological examination is warranted. Further, we found a substantial complication rate after breast reconstruction, which mainly concerned late cosmetic issues, almost always leading to additional surgery. In this respect, patients should be informed preoperatively that an optimal cosmetic effect cannot unconditionally be achieved in just one single operation. Concerning the complication rate after BR, we did not find a significant difference between affected and unaffected women. In our opinion, our data are providing additional data on this issue and may help to inform women considering prophylactic mastectomy and their physicians, in the complex process of decision-making.

mastectomy
brca1/2
breast cancer
complications
prevention
unexpected carcinomas


Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes

ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 μM. The OGD-induced ATP release was inhibited by Gd3+ and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl− channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X7 receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd3+ and arachidonic acid. The channel was found to be permeable to ATP4− with a permeability ratio of PATP/PCl = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions.
Introduction
Astrocytes do not merely support, but actively regulate, neuronal functions by forming highly elaborate networks with other astrocytes and neurons [1–4]. Astrocytes communicate with neurons by releasing extracellular signaling molecules such as ATP and glutamate, while they communicate with each other via gap junctions, in addition to these gliotransmitters. Astrocytic ATP release has been shown to be induced in response to receptor stimulation by UTP [5], glutamate [6], and noradrenaline [7], to mechanical stimulation [8–10], to osmotic swelling [11], and to exposure to low Ca2+ solution [8, 9, 12]. Although ischemic stress is known to induce ATP release from rat hippocampal slices [13], it is not known whether astrocytes respond to ischemia with ATP release. The first purpose of the present study was to address this question.
Astrocytes express maxi-anion channels with a large single-channel conductance of 200–400 pS [14–16]. Recently, we have demonstrated that astrocytic maxi-anion channels are activated by chemical ischemia and serve as one of the major pathways for glutamate release [17]. The maxi-anion channel was found to possess a pore with a size large enough to permeate ATP [18] and was found to actually conduct anionic forms of ATP in mammary C127 cells under hypotonic conditions [19], in kidney macula densa cells in response to increased luminal NaCl concentrations [20], and in cardiac myocytes under hypotonic or ischemic conditions [21]. Thus, there is a possibility that maxi-anion channels serve as one of the pathways for ATP release from astrocytes under ischemic conditions. The second purpose of this study was to test this possibility.
Materials and methods
Cells
Astrocytes were obtained from neonatal mouse brain cortex and cultured, as previously described [17, 22], in Eagle’s MEM supplemented with L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% FBS, and 2.2 mg/ml NaHCO3.
HEK293 cells stably transfected with recombinant P2X2 purinergic receptors were prepared and cultured, as described elsewhere [23]. The HEK-P2X2 cells were used after dissociation into single cells and culturing in suspension for over 10 min.
Solutions and chemicals
The standard Ringer solution contained (mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 Na-HEPES, 6 HEPES, and 5 glucose (pH 7.4, 290 mosmol/kg H2O). For inside-out experiments, we used standard Ringer solution both in the bath and in the pipette. The pipette solution for biosensor whole-cell experiments contained (mM): 125 CsCl, 2 CaCl2, 1 MgCl2, 10 EGTA, and 5 HEPES (pCa 7.6, 275 mosmol/kg H2O, pH 7.4 adjusted with CsOH). For ATP conductance measurements in the inside-out configuration, the bath solution was substituted with a solution of 100 mM Na4ATP.
Oxygen-glucose deprivation (OGD) stress was applied by switching the perfusate from the standard Ringer solution to the OGD solution. The OGD solution was prepared by replacing D-glucose in Ringer solution with 2-deoxyglucose and was continuously bubbled with 100% argon gas for more than 1 h before and during experiments. The oxygen concentration (pO2) measured using an oxygen sensor (LICOX A3R, GMS, Kiel-Mielkendorf, Germany) was 118.4 ± 1.1 mmHg (n = 8) for standard Ringer solution exposed to air in the experimental perfusion chamber. Under OGD conditions, the oxygen concentration in the perfusate solution decreased to a stable value of 7.5 ± 0.6 mmHg (n = 9) at the inlet and 40.1 ± 1.8 mmHg (n = 8) at the outlet of the experimental patch-clamp chamber.
GdCl3 was stored as a 50-mM stock solution in water and added directly to solutions immediately before each experiment. Arachidonic acid, phloretin, brefeldin A (BFA), 1-octanol, probenecid, glibenclamide, and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, USA) and BAPTA-AM from DOJINDO (Kumamoto, Japan); they were added to solutions immediately before use from stock solutions in DMSO. DMSO did not have any effect when added alone at concentrations employed in the present study (less than 0.1%).
Osmolality of all solutions was measured using a freezing-point depression osmometer (OM802, Vogel, Kevelaer, Germany).
Luciferin-luciferase ATP assay
The bulk extracellular ATP concentration was measured by a luciferin-luciferase assay (ATP Luminescence Kit; AF-2L1, DKK-TOA, Tokyo, Japan) with an ATP analyzer (AF-100, DKK-TOA, Tokyo, Japan), as previously described [19, 21, 24], using astrocytes cultured in 12- or 24-well plates. After the culture medium was totally replaced with isotonic Ringer solution (1,000 and 425 μl for 12- and 24-well plates, respectively), cells were incubated for 60 min at 37°C. An aliquot (100 μl) of the extracellular solution was collected as a control sample for background ATP release. An OGD challenge was then applied by gently removing most of the remaining extracellular solution (875 and 300 μl for 12- and 24-well plates, respectively) and adding the OGD solution (1,000 and 400 μl for 12- and 24-well plates, respectively), and then placing the plates in an airtight incubator where normal air was replaced with 100% argon. At the specified time points, the plate was carefully rocked again to ensure homogeneity of the extracellular solution, and samples (20 and 50 μl for 12- and 24-well plates, respectively) were collected from each well for the luminometric ATP assay. The ATP concentration was measured by mixing 20 or 50 μl of sample solution with 530 or 500 μl normal Ringer solution and 50 μl of luciferin-luciferase reagent. At this ratio, the ionic salt sensitivity of the luciferase reaction was negligible. When required, drugs were added to the OGD solution to give the final concentrations as indicated. Since Gd3+ is known to interfere with the luciferin-luciferase reaction [25], we supplemented the luciferin-luciferase assay mixture with 600 μM EDTA when the samples contained Gd3+. Other drugs employed in the present study had no significant effect on the luciferin-luciferase reaction.
Biosensor ATP measurements
To measure the local concentration of ATP released from single astrocytes, the biosensor technique was employed, as described previously [23]. ATP-dependent changes in whole-cell P2X2 receptor currents were recorded from a biosensor HEK-P2X2 cell at a holding potential of −50 mV. Before the actual measurements of ATP release, a calibration curve was constructed by recording the biosensor responses to known concentrations of ATP. The biosensor cell in the whole-cell configuration was lifted by means of a micromanipulator and positioned near a handmade local microperfusion device consisting of several inlet tubes. Each of the inlet tubes was filled with a different concentration of ATP solution ranging from 1 to 20 μM. To minimize the effect of desensitization, the sensor cell was exposed to ATP puffed every 3 min for 1 s to a maximum of five applications. During the measurements of ATP from an individual astrocyte under OGD stress, the biosensor cell was gently touched to the surface of the target cell. From the recorded currents, the concentration of ATP was calculated according to the calibration curve.
Patch-clamp experiments and data analysis
Patch-clamp experiments were performed, as described previously [17, 19], using an EPC-9 patch-clamp system (HEKA Electronics, Lambrecht/Pfalz, Germany). The membrane potential was controlled by shifting the pipette potential (Vp). Currents were sampled at 3 kHz and filtered at 1 kHz. Data acquisition and analysis were done using Pulse+PulseFit (HEKA Electronics, Lambrecht/Pfalz, Germany). Whenever the bath Cl− concentration was altered, a salt bridge containing 3 M KCl in 2% agarose was used to minimize variations of the bath electrode potential. Liquid junction potentials were corrected online. All experiments were performed at room temperature (23–25°C).
Single-channel amplitudes were measured by manually placing a cursor at the open and closed channel levels. Background currents were subtracted and the mean patch currents were measured at the beginning (first 25–30 ms) of current traces in order to minimize the contribution of voltage-dependent current inactivation. The reversal potentials were calculated by fitting I–V curves to a second-order polynomial [18] or measured directly from ramp currents. Permeability ratios were calculated, as described previously [19, 21]. Data were analyzed in Origin 6 or 7 (OriginLab Corporation, Northampton, MA, USA). Pooled data are given as means ± SEM of n observations.
Statistical differences of the data were evaluated by analysis of variance (ANOVA) and the paired or unpaired Student’s t-test where appropriate and were considered significant at P < 0.05.
Results
OGD induces massive release of ATP from astrocytes
Under control conditions, when astrocytes were incubated in standard Ringer solution, the basal release of ATP was very low and did not exceed 0.25 nM over 40 min incubation time (Fig. 1a: open circles). In contrast, when cells were transferred to OGD conditions, the bulk ATP in the incubation medium rapidly increased from the basal level to a peak level of approximately 2 nM within 15–20 min. After this period, the extracellular ATP concentration remained high up to 30 min of incubation and then slightly decreased at 40 min of incubation under OGD stress (Fig. 1a: filled circles).
Fig. 1OGD-induced ATP release from astrocytes detected by the luciferin-luciferase assay. a Time course of ATP release during incubation with control standard Ringer solution (open circles) and with the OGD solution (filled circles). b Pharmacological profiles of ATP release measured 20 min after application of OGD solution. Each data point or column represents the mean ± SEM (vertical bar). *Significantly different from the control OGD value in the absence of drugs at P < 0.05
In the following pharmacological experiments, we used an incubation time point of 20 min, when the net ATP release induced by OGD reached a steady-state level. Application of extracellular 50 μM Gd3+ nearly completely inhibited OGD-induced ATP release from cultured mouse astrocytes. Arachidonic acid (20 μM) also significantly inhibited OGD-induced ATP release. In contrast, OGD-induced ATP release was not significantly affected by blockers of the volume-sensitive outwardly rectifying (VSOR) Cl− channel, phloretin (100 μM) and glibenclamide (200 μM), of the CFTR Cl− channel, glibenclamide (200 μM), and of the transporter MRP, probenecid (1 mM). Also, the ATP release was not sensitive to inhibitors of connexin or pannexin hemichannels, 1-octanol (2 mM) and carbenoxolone (100 μM), and a blocker of the P2X7 receptor, brilliant blue G (1 μM). Preincubation of the cells with an inhibitor of vesicular transport, brefeldin A (BFA, 5 μM), for 120 min and together with an intracellular Ca2+ chelator, BAPTA-AM (5 μM), for 30 min had no significant effect on the OGD-induced release of ATP (Fig. 1b).
In order to detect ATP release at the surface of single astrocytes, we employed a biosensor ATP assay technique [23]. We first established the calibration curve by recording the responses of biosensor HEK-P2X2 cells to different known concentrations of ATP (Fig. 2a). We then gently put the biosensor cell close to the surface of a target astrocyte and recorded the whole-cell current responses from the biosensor cell. As shown in Fig. 2b (top panel), virtually no ATP-evoked currents were observed during perfusion of standard Ringer solution. However, inward current spikes were consistently recorded upon perfusion with OGD solution after a lag period of 7.2 ± 1.0 min (n = 5) (Fig. 2b: middle panel). The OGD-induced inward current responses were eliminated by application of an ATP-hydrolyzing enzyme, apyrase (Fig. 2b: bottom panel). OGD-induced responses were never observed in biosensor cells alone which were positioned remote from target astrocytes. Using the calibration curve, the local ATP concentration was estimated to be 3.7 ± 0.1 μM at the surface of single astrocytes subjected to OGD stress.
Fig. 2Cell surface ATP release induced by OGD from single astrocytes. a Calibration of ATP measurements by the biosensor technique. Top panel: representative current responses evoked by puff applications of 1–20 μM ATP in a biosensor HEK-P2X2 cell under whole-cell clamp. Bottom panel: concentration-response curve of the ATP-induced currents recorded from biosensor cells. Each data point represents the mean ± SEM (vertical bar). b Representative current traces recorded from biosensor cells in close proximity to target astrocytes after perfusion with standard Ringer solution (top panel) or with the OGD solution in the absence (middle panel) or presence (bottompanel) of 0.1 mg/ml apyrase, which was applied after current responses appeared. When apyrase was applied from the beginning of OGD application, no current response was observed in biosensor cells positioned next to astrocytes (data not shown)
From these data, it is concluded that in response to OGD stress, Gd3+- and arachidonic acid-sensitive ATP release from astrocytes is induced, increasing the extracellular ATP concentration to a level high enough to stimulate most types of purinergic receptors.
OGD activates ATP-conductive maxi-anion channels in astrocytes
The fact that both Gd3+ and arachidonic acid are potent blockers of maxi-anion channels in mouse astrocytes [17] suggests that OGD-induced ATP release involves the activity of maxi-anion channels. In cell-attached experiments, OGD stress actually induced activation of anion channels with a large conductance (Fig. 3a), as seen to occur in a similar manner upon stimulation by hypotonicity or chemical ischemia in mouse astrocytes [17]. The unitary I–V relationship for these events exhibited slight outward rectification, and the mean slope conductance was 427 ± 23 pS at +60 mV and 262 ± 21 pS at −50 mV (Fig. 3b). In cell-attached patches, OGD-induced activation of the maxi-anion channel started with a lag time of several minutes and reached its peak within 10 min (8.8 ± 1.2 min, n = 6), as shown in Fig. 3c.
Fig. 3OGD-induced single-channel activity of the maxi-anion channel in cell-attached patches on astrocytes. a Representative current trace recorded during application of alternating pulses (the protocol shown in inset above). Note that voltage-dependent inactivation of channel activity took place shortly after application of step pulses to ± 50 mV. b Current-voltage (I–V) relationship for unitary events of the maxi-anion channel. The reversal potential was −4.6 ± 1.9 mV. Each data point represents the mean ± SEM (vertical bar). c Time course of OGD-induced activation of the maxi-anion channel. The mean patch currents recorded at ±50 mV are plotted as a function of time
The single-channel activity induced by OGD persisted after excision of the patch (Fig. 4: control). In the inside-out mode, however, the channel activity was blocked by extracellular Gd3+ (50 μM) in backfilled pipettes and by intracellular (bath) application of 20 μM arachidonic acid (Fig. 4: Gd3+ and arachidonate). In contrast, phloretin (100 μM) failed to affect the maxi-anion channel activity (Fig. 4: phloretin).
Fig. 4Pharmacological profile of OGD-activated maxi-anion channel events in inside-out patches excised from astrocytes. a Representative current traces recorded during application of alternating pulses (the protocol shown in inset above) in the absence (control) and presence of Gd3+ (50 μM in the pipette), arachidonic acid (20 μM in the bath), and phloretin (100 μM in the bath). b Effects of Gd3+, arachidonic acid, and phloretin on the OGD-induced single-channel activity recorded at ± 25 mV. Data are normalized to the mean currents before application of drugs. Each column represents the mean ± SEM (vertical bar). *Significantly different from the control value at P < 0.05
In inside-out patches, when the bath solution was changed from standard Ringer solution to a 100 mM Na4ATP solution, the reversal potential shifted from 0 mV to −20.6 ± 0.9 mV (n = 6); from this result, a PATP/PCl value was estimated to be 0.11 ± 0.01 (Fig. 5).
Fig. 5Representative ramp I–V records of single-channel events of the maxi-anion channel preactivated by OGD in an inside-out patch exposed to standard Ringer solution (chloride) and one exposed to 100 mM Na4ATP solution (ATP). The inward currents recorded in the ATP-exposed patch represent ATP4− currents. The amplitude of outward currents became smaller when ATP4− was present in the bath, suggesting a blocking of currents by ATP4−, as observed in mammary C127 cells [19]
These results indicate that in astrocytes OGD stress activates a maxi-anion channel which can conduct anionic forms of ATP and is sensitive to Gd3+ and arachidonic acid.
Discussion
In the brain, bidirectional communication takes place between glial cells and neurons. Astrocytes clear synaptically released glutamate from the synaptic cleft, receive signals from neurons at certain receptors, and release molecules, called gliotransmitters, that modulate synaptic transmission, protect neurons from impairment, or repair neuronal tissues after damage. ATP and glutamate represent major gliotransmitters. It has been reported that astrocytes release ATP in response to a number of stimuli, including receptor activation [5–7], mechanical or osmotic perturbation [8–11], and deprivation of extracellular Ca2+ [8, 9, 12]. The present study demonstrated that astrocytes also respond to ischemic stress mimicked by OGD with ATP release (Figs. 1a and 2b). ATP released from astrocytes has been shown to regulate glutamatergic synaptic transmission [6, 7, 10], inhibit excitability of retinal neurons [26], activate microglia [27, 28], modulate some functions of astrocytes [11], and protect astrocytes themselves from H2O2-induced cell death [29]. Pathophysiological functions of ischemia-induced ATP release from astrocytes remain to be elucidated.
There has been much controversy about the pathway of nonlytic release of ATP from a variety of cell types [30, 31]. The candidate pathways proposed for astrocytic ATP release include connexin hemichannels [8], MRP [11], P2X7 receptors [12], and exocytotic vesicular transport [9]. In the present study, however, OGD-induced ATP release was shown to be insensitive to blocking agents for connexin hemichannels (1-octanol, carbenoxolone), MRP (probenecid), P2X7 receptors (brilliant blue G), and exocytosis (BFA plus BAPTA-AM) (Fig. 1b). Recently, it was reported that pannexins form ATP-conductive channels when expressed in Xenopus oocytes [32]. However, a possible involvement of the pannexin hemichannel in OGD-induced ATP release from astrocytes can be ruled out by the present observation (Fig. 1b) of insensitivity to carbenoxolone, which is a potent blocker of not only connexin but also pannexin hemichannels as well [33]. Although VSOR anion channels were shown to be permeable to ATP at large positive potentials in endothelial cells [34], potent VSOR blockers (phloretin, glibenclamide) failed to affect OGD-induced ATP release from astrocytes in the present study (Fig. 1b). In contrast, Gd3+ and arachidonic acid were found to prominently suppress ATP release from mouse astrocytes subjected to OGD (Fig. 1b). Our previous study [17] showed that in mouse astrocytes, maxi-anion channels are activated by chemical ischemia and are sensitive to both Gd3+ and arachidonic acid. The present study demonstrated that maxi-anion channels are activated by OGD (Fig. 3) and that OGD-activated maxi-anion channels are sensitive to Gd3+ and arachidonic acid (Fig. 4) and can conduct anionic forms of ATP (Fig. 5). On balance, it appears that the maxi-anion channel serves as a major pathway for OGD-induced ATP release from mouse astrocytes in primary culture. Our preliminary study has shown that in mouse hippocampal slices, astrocytes also respond to OGD with the activation of maxi-anion channels (H.-T. Liu, R.Z. Sabirov, Y. Okada, unpublished observations). Since ischemia-like stress was reported to induce ATP release from hippocampal slices [13], it is highly possible that the maxi-anion channel also provides a pathway for ischemia-induced ATP release from astrocytes in slice preparations.

oxygen-glucose deprivation
atp release
maxi-anion channel
astrocytes