Isolated Limb Perfusion with Tumor Necrosis Factor Alpha and Melphalan for Locally Advanced Soft Tissue Sarcoma: Three Time Periods at Risk for Amputation

Background The aim of this study was to investigate the long-term limb salvage rate and overall survival after isolated limb perfusion (ILP) with tumor necrosis factor alpha and melphalan for locally advanced soft tissue sarcoma (STS).
Limb salvage in patients with locally advanced extremity soft tissue sarcoma (STS) continues to be a challenge. Survival in these patients is determined by the development of distant metastases and is not improved with the amputation of the affected limb.1,2 Besides amputation, an extensive surgical procedure followed by radiotherapy is a treatment option.3 Rosenberg et al.1 showed the same disease-free and overall survival as amputation in the early 1980s with this treatment regimen. Preoperative therapies to improve limb salvage rates have been proposed. Suit et al.4 reported in 1981 on the use of preoperative radiotherapy. Eilber et al.2,5 combined preoperative (intra-arterial or systemic) chemotherapy and radiotherapy to improve resectability rates. In a randomized trial, O’Sullivan et al.6 reported a greater risk of wound complications in the preoperative radiotherapy group compared with the postoperative radiotherapy group. The use of brachytherapy may also improve local control and avoid amputation.7 The current treatment strategy of high-grade limb sarcomas is wide local resection, with the goal of achieving a R0 resection with a 2-cm margin. If the margin is < 2 cm or if a R1 resection (microscopically involved margin) is performed, adjuvant radiotherapy with 50–70 Gy is indicated to reduce the risk of local failure.3 The question whether radiotherapy should be given before or after surgery is still unanswered.6
Another strategy for limb salvage in locally advanced extremity STS is to perform an isolated limb perfusion (ILP) with cytostatic agents. This procedure, which was originally developed for the treatment of melanoma of the limb in 1957, was also applied to the treatment of STS of the limb. In their first experience, Krementz et al.8 showed an early response rate of 83% with melphalan alone; however, complete regression of the tumor was rarely seen. Other perfusion agents in the treatment of limb STS were therefore investigated. Rossi et al.9 claimed that doxorubicin was efficacious; another study10 showed that doxorubicin alone was ineffective and that combined with melphalan, it was too toxic. Cisplatin also proved to be less effective than melphalan in the limb perfusion setting of sarcomas, and carboplatin was too neurotoxic.11–13
With the addition of tumor necrosis factor alpha (TNF-α) to the perfusion circuit, Lienard et al.14 made a step forward in the treatment of locally advanced extremity STS. A large European multicenter study proved the ILP concept in the limb salvage procedures for locally advanced STS with TNF-α and melphalan. The objective response rate was 75%, and a limb salvage rate of 82% was achieved with minimal treatment-related morbidity.15 Since 1991, patients with locally advanced STS of the limbs have been treated at the University Medical Center Groningen by ILP with TNF-α and melphalan with or without interferon gamma as perfusion agents, followed by delayed surgical excision and postoperative radiotherapy if a marginal resection or nonradical resection was performed. Recently we encountered long-term local morbidity, and therefore the aim of the present study was to analyze the limb salvage rate and survival in patients with locally advanced STS of the extremities that were treated in our center and to report the late effects of this treatment modality.
PATIENTS AND METHODS
Patient Characteristics
During 1991–2003, a total of 73 patients with STS of the extremity underwent 77 perfusions with a combination of TNF-α and melphalan, with (n = 19) or without (n = 58) interferon gamma. Thirty-six men and 37 women with a median age of 54 (range 14–80) years were treated. Tumors were considered unresectable because of size, their multicentricity in the limb, or fixation to the neurovascular bundle and/or bone, and therefore amputation was the only treatment option. Perfusion was performed at the iliac level in 32 cases (42%), at the popliteal level in 23 cases (30%), and at the femoral and axillary level in 11 cases each (14%). There were 60 primary (82%) and 13 recurrent (18%) sarcomas. Sixty-two sarcomas were located in the leg (85%) and 11 were located in the arm (15%). All patients were treated after informed consent was obtained according to institutional guidelines. Nineteen different histological types of STS were distinguished. The pathological grade of the tumor was scored following the criteria of Coindre et al.,17 and the stage of the tumor was scored according to the American Joint Committee on Cancer (AJCC) criteria16 (Table 1). Median tumor size was 16.2 (range 8.3–23) cm. In case of multifocal disease, the largest diameter was used.
TABLE 1.Histological grade and stage of tumorsaCharacteristicn%Grade  I1014  II2332  III4054Stage  I1014  II11  III5069  IV1216a Grade according to Coindre et al.,17 and stage according to American Joint Committee on Cancer.16
Perfusion Technique
The perfusion technique used at the University Medical Center Groningen is based on the technique developed by Creech et al.18 and described elsewhere.19 The major modifications during the last 30 years were the use of modern thermal blankets, improvement in leakage monitoring, and the introduction of a membrane oxygenator and heat exchanger to ensure optimal perfusion at 39–40°C. Because extensive washing with 6 L of saline is used, systemic inflammatory response syndrome (SIRS) is rarely seen.20 After surgery, patients can be monitored on the recovery ward instead of the intensive care unit.
Assessment of Tumor Response, Tumor Remnant, and Follow-up
Responses were assessed by standardized World Health Organization criteria and on the basis of physical examination and/or imaging investigations (magnetic resonance imaging and computed tomographic scans).21 Complete response was defined as the disappearance of all measurable disease in the limb for > 4 weeks, partial response as regression of the tumor size by > 50% of the largest diameter for > 4 weeks, and no change as regression of < 50% of the tumor in the limb or progression of < 25% for longer than 4 weeks. Resection of the tumor remnants was performed 2–15 weeks (median, 8 weeks) after perfusion. After resection, response was also assessed by pathological examination. The tumor remnants were measured in three dimensions and the percentage of necrosis estimated in relation to the complete tumor volume. Representative tumor sections were taken, encompassing macroscopically different tumor areas, including necrosis. As a general rule, one section per centimeter largest diameter with a minimum of three was taken. On the basis of an integration of gross and microscopic findings, a final estimate of the percentages of viable and necrotic or regressive tumor was made.
Excision margins were also evaluated on pathological examination and classified as radical when the resection margins were free of tumor cells (complete resection, R0), as R1 when resection margins were microscopically involved, or as R2 when resection margins were macroscopically positive involved. Postoperative radiotherapy (60–70 Gy) was considered indicated in case of < 95% necrosis on pathological examination of the tumor or with marginal or microscopically positive resection margins. All patients were followed after perfusion treatment in a standardized protocol. Median follow-up was 27 (range 2–138) months.
Statistical Analysis
Survival and limb salvage curves were calculated according to the Kaplan-Meier method and log rank test.22 Values of P < 0.05 were considered to be statistically significant. GraphPad Prism version 2.0 for Windows statistical software was used.
RESULTS
Tumor Response
A clinical complete response was observed after 19 ILPs (25%), a partial response after 53 ILPs (69%), and no change after 5 ILPs (6%); local progression was never observed. Resection of the remnant tumor was performed in 68 patients (93%). The pathological response is illustrated in Fig. 1. After 17 ILPs (23%), no viable tumor cells were found on pathological examination. In 29 ILPs (37%), ≥ 90% necrosis was found on pathological examination. When both groups were added together, a good response to ILP was found in 60% of the patients. In 17 ILPs (22%), an intermediate response was found on pathological examination (necrosis 50%–80%). After eight ILPs (10%), < 20% of necrosis or no necrosis was found on pathological examination. In five patients (7%), tumor response was not assessed because of progression of distant metastases in four patients and a local recurrence in one patient that necessitated a second perfusion, resulting in 90% necrosis of the tumor. No correlation could be demonstrated between grade and percentage of tumor necrosis after perfusion (Pearson’s correlation). Postoperative radiotherapy (total dose 60–70 Gy, 25 × 2 Gy daily and 10–20 Gy boost) was given in 37 patients with microscopically involved or marginally free resection margins. Radiotherapy was initiated within 5–6 weeks after tumor resection. Radiation treatment was delivered through a multiple-field technique with computed tomographic treatment planning on a linear accelerator, 6–15 MV.
FIG. 1.Percentage of necrosis estimated at pathological examination of resected tumor remnant in relation to number of patients.
Amputations and Limb Salvage
A total of 21 amputations (28%) were performed. Table 2 presents the time interval between ILP and amputation and the rationale for amputation. Overall 1, 5, and 10 years’ limb salvage was 80.1% ± 4.8%, 68.2% ± 6.5%, and 60.6% ± 9.2%, respectively (Fig. 2). When we analyzed the limb salvage curve, we found that the risk for amputation could be divided into three time episodes. The first period occurs within the first year or year and a half after perfusion (n = 17 patients), with amputation performed to treat massive perfusion-induced necrosis of the tumor and overlying skin, which resulted in a soft tissue deficit (n = 6); tumor recurrence after perfusion (n = 5); wound complications after ILP followed by radiotherapy (n = 2); a microscopically involved resection margin with the rejection of the patient for adjuvant radiotherapy of the foot (n = 2); insufficient clinical response (one patient); and arterial thrombosis with no vascular reconstruction possibilities 2 months after resection of a local recurrence in the groin (n = 1).
TABLE 2.Amputations performed in 21 patients according to interval durationPatient no.DiagnosisAge (y)Interval (mo)ResectionRTRationale for amputationCurrent status1PUS600R0NoPostperfusion necrosis38NED 120 months2Angiosarcoma741R2NoLocal recurrenceDOD 11 months3Fibrosarcoma761R0NoPostperfusion necrosisNED 2 months4PUS672R0NoPostperfusion necrosisDOD 9 months5Epithelioid Sarcoma212R0NoPostperfusion necrosisDOD 54 months6Leiomyosarcoma172R0NoInsufficient clinical responseDOD 7 months7Liposarcoma602R1NoR1 resection, RT not possibleAWD 10 months8PNET623R0NoLocal recurrenceDOD 17 months9Synovial sarcoma393R0NoPostperfusion necrosisDOD 50 months10PUS633R1NoPostperfusion necrosisNED 72 months11Angiosarcoma804R1NoLocal recurrenceDOD 10 months12Synovial sarcoma654R1NoR1 resection, RT not possibleNED 6 months13Epithelioid Sarcoma226R2NoLocal recurrenceaDOD 39 months14Hemangioma pericytoma508R0YesWound complications after ILP with radiotherapyaAWD 65 months15PUS7112R0YesWound complications after ILP with radiotherapyaNED 14 months16PUS6115R0YesArterial occlusionAWD 17 months17Synoviosarcoma4218R1YesLocal recurrenceNED 20 months18Liposarcoma5337R0YesLocal recurrenceDOD 110 months19Liposarcoma3958R0NoLocal recurrenceDOD 120 months20PNET56110R0YesCritical leg ischemiaNED 118 months21Chondrosarcoma18125R0YesCritical leg ischemiaNED 134 monthsPUS, pleomorphic undifferentiated sarcoma; RT, radiotherapy; NED, no evidence of disease; AWD, alive with disease; DOD, death of disease; PNET, malignant peripheral nerve sheath tumor; R1 resection, microscopically involved resection margin; ILP, isolated limb perfusion.a Patients treated for second ILP with tumor necrosis factor alpha and melphalan.FIG. 2.Limb salvage curve in patients with locally advanced soft tissue sarcoma treated with tumor necrosis factor alpha, melphalan, and isolated limb perfusion.
The second time period was within 5 years after ILP, with two amputations performed for late local recurrent disease (37 and 58 months after perfusion). The third episode occurred approximately 10 years after perfusion. Amputation was performed for critical leg ischemia with neuropathy due to treatment-induced atherosclerosis of the remaining tibial artery that was not suitable for arterial reconstruction (110 and 125 months after perfusion). An example of the clinical appearance of patient 21 is shown in Fig. 3. In this patient, a popliteal ILP was performed at the age of 18 years for a chondrosarcoma. After marginal resection, this patient received 66 Gy adjuvant radiotherapy. Ten years after ILP, an amputation was performed because of critical leg ischemia. No recurrent disease was found on pathological examination of the amputated specimen. Another two patients developed a pathological fracture of the femur due to radiation-induced osteonecrosis (78 and 129 months after perfusion). These four patients with late post-ILP complications received high-dose postperfusion radiotherapy (60–70 Gy).FIG. 3.Clinical appearance of lower leg of patient 21 (Table 3) before amputation for critical leg ischemia.
Systemic Metastases and Survival
Twelve patients presented with distant metastases at time of ILP (16% stage IV AJCC); half of these patients had lung metastases, and the other half had lymph node metastases. Eleven of these patients died of disease after a median period of 9 (range 2–54) months; one patient is alive with no evidence of disease after 11 months. During follow-up, 25 patients (36%) developed distant metastases at a median interval of 9 (range 2–100) months. A statistically significant difference (P < .001) was observed between patients with no distant metastases at the time of ILP compared with patients with metastases at the time of ILP (Fig. 4). Overall 1, 5, and 10 years’ survival for all patients was 82.9% ± 9.2%, 58.7% ± 13.1%, and 42.5% ± 18.2%, respectively (Fig. 4).
FIG. 4.Overall survival in patients with locally advanced soft tissue sarcoma treated with isolated limb perfusion (ILP), tumor necrosis factor alpha, and melphalan. Statistically significant difference was observed between patients with no distant metastases at time of ILP (mets −) compared with patients with metastases at time of ILP (mets +).
DISCUSSION
The results of a European multicenter trial performed in the 1990s led to the approval of the use of TNF-α for ILP in patients with locally advanced extremity sarcomas by the European Medicine Evaluation Agency.15 ILP with TNF-α is currently available in more than 30 centers, and in 2002, a total of 350 so-called TNF-α perfusions were performed. As one of the first centers that participated in the TNF-α ILP experience, and with more than a decade’s experience, we recently encountered long-term treatment-related morbidity necessitating amputation of the perfused limb 10 years after treatment. For this reason, we analyzed our results of ILP with TNF-α and melphalan and describe our results in the present study.
We observed an overall response rate of 82%, which is in the range of the 63%–91% response rates reported in the literature.15,23,25 Although a suggestion has been made for a relation between the grade of sarcoma and the response to TNF-α ILP, we could not demonstrate a correlation between grade and the percentage of necrosis after ILP with TNF-α. This is in concordance with the results of the Amsterdam group.23 Various reports have shown that a limb salvage rate of 81%–86% can be achieved in patients with locally advanced limb sarcoma.15,23,25 An independent review committee reconsidered the unresectability criteria of all patients enrolled onto the European study. Eighty percent of the patients in this study met the criteria for unresectability, and survival curves based on a match-control study with cases of the Scandinavian Soft Tissue Sarcoma Databank showed that TNF-α ILP had no negative effect on survival.26
We used the Kaplan-Meier method to calculate limb salvage because this method adjusts for censored observations, i.e., patients who were alive and well at the time of last contact or patients who died of distant metastases but who retained preserved limb function. By use of this method, we calculated a 1-year limb salvage rate of 80%. Amputations were performed mostly because of postperfusion-related complications or early local recurrence in the first year after ILP. A second curve in limb salvage was observed within 5 years after TNF-α ILP in two patients with late local recurrences. A third bend in the limb salvage curve was observed approximately 10 years after ILP. This was a new observation in two patients who had critical leg ischemia with ulceration and continuous pain. Besides ILP with TNF-α and melphalan, both patients received adjuvant radiotherapy (66 and 70 Gy) after marginal tumor resection.
What seems to be the cause of this late morbidity? Analysis of the functional and long-term morbidity in 97 patients with stage I melanoma treated with ILP with melphalan as the sole perfusion agent in our center showed after a median follow-up of 36 (range 12–76) months, no patients had critical leg ischemia.27 The Rotterdam and Amsterdam perfusion group reported long-term morbidity consisting of muscle atrophy or fibrosis in 11% of the patients after ILP with melphalan; however, cases of critical leg ischemia are not described.28 The fact that in our series no muscle atrophy or fibrosis was found might be explained by the fact that we always perform a lateral fasciotomy after ILP to prevent a compartment syndrome. When we performed a literature search for late morbidity after ILP with TNF-α and melphalan, no studies could be retrieved.
The clinical importance of late morbidity after radiotherapy has evolved since Eifel et al.29 retrospectively reviewed the medical records of 1784 FIGO stage IB patients receiving primary radiotherapy at the M. D. Anderson Cancer Center between 1960 and 1989. She showed that after 5 years, there was a small but continuous risk of experiencing major complications of radiotherapy (i.e., urinary, rectal, and small bowel complications) at up to 20 years of follow-up. Johansson et al.30 described a high occurrence of severe neuropathy closely linked to the development of fibrosis around the nerve trunks after aggressive postoperative telecobalt therapy received in 1963–1965 in a group of 71 patients with breast cancer who were initially treated with modified radical mastectomy. Radiotherapy damage to the vascular system was demonstrated by Hopewell31 in an experimental setting; arteries of hamster cheek pouch showed localized constrictions after irradiation. These constrictions were caused by clones of endothelial cells and may be the predominant factor influencing the degeneration of the capillary bed after radiotherapy.32 Evidence of this occlusive effect of vessels by proliferating endothelial cells after radiation have also been reported by other investigators.33
Another argument to explain the observed late morbidity, at least in part, to radiotherapy is the fact that another two patients who received adjuvant radiotherapy after ILP with TNF-α and melphalan developed a pathological fracture of the femur (78 and 129 months after ILP with TNF-α and melphalan). Radiotherapy-induced osteonecrosis is a well-known phenomenon. Lin et al.34 described 12 fractures of the femur after surgery and irradiation for STS of the thigh. Treatment of these fractures was difficult and demanding, with only four bony unions after a mean follow-up of 37 months. When we add up the evidence of developing fibrosis after ILP with melphalan and the development of fibrosis after radiotherapy, the combination of the two regimens could explain the observed late morbidity rate in the present series.
Overall survival for all patients showed a steadily decline, with a 10-year percentage of 42%. Even after 110 and 120 months, patients die of distant metastases. Sixteen percent of the patients had metastases at the time of ILP. A marked difference in survival was observed when comparing patients with pulmonary or lymph node metastases with patients lacking metastases at time of ILP. Five-year overall survival of 59% in this series is higher than the reported 5-year survival of 48% in the Amsterdam experience23 and the reported 32% of Lejeune et al.24 This is an unexpected observation because selection criteria for ILP with TNF-α between the institutes are comparable. High-grade tumors and tumors > 5 cm in diameter have a great tendency to metastasize. Patients with such tumors could theoretically benefit from neoadjuvant chemotherapy. A quantitative meta-analysis of data from 14 trials of doxorubicin-based adjuvant chemotherapy showed a benefit from systemic adjuvant chemotherapy of 6% for local relapse-free interval, but there was no overall survival benefit at 10 years.35 Delaney et al.36 developed a regimen of preoperative chemotherapy consisting of mesna, Adriamycin, ifosfamide, and dacarbazine (MAID) interspersed with radiotherapy, followed by resection and postoperative chemotherapy with or without radiotherapy to improve outcome in patients with high-grade extremity STS. Compared with a historical group of control patients, outcome in the MAID group was better. In an update of 64 patients, 5 required amputation because of disease, 3 had unresectable disease, and 1 patient refused surgery. Estimated 3-year survival and local-regional control were 75.1% and 79.3%, respectively. These results are comparable with the results of the present study.37 However, systemic therapy is associated with systemic toxic effects, in contrast with the mild systemic side effects observed after ILP with TNF-α and melphalan.
Since 1992, we have not changed the indication for TNF-α perfusion. Patients who were candidates for amputation of the involved limb, as assessed by preoperative magnetic resonance imaging, were offered an ILP with TNF-α and melphalan, with the goal of preserving the limb with a locally advanced STS. After ILP patients received a delayed surgical resection, adjuvant radiotherapy was provided to patients with marginal or microscopically positive resection margins. This treatment resulted in a high limb salvage rate in patients with locally advanced STS, although late morbidity can occur, especially when adjuvant postoperative radiotherapy is applied. Therefore, continuous follow-up of these patients is warranted.

isolated limb perfusion
tumor necrosis factor alpha
melphalan
sarcoma
amputation
radiotherapy
late morbidity


Cerebral blood volume, genotype and chemosensitivity in oligodendroglial tumours

Introduction The biological factors responsible for differential chemoresponsiveness in oligodendroglial tumours with or without the −1p/−19q genotype are unknown, but tumour vascularity may contribute. We aimed to determine whether dynamic susceptibility contrast (DSC) magnetic resonance imaging (MRI) could distinguish molecular subtypes of oligodendroglial tumour, and examined the relationship between relative cerebral blood volume (rCBV) and outcome following procarbazine, lomustine and vincristine (PCV) chemotherapy.
Introduction
Oligodendroglial tumours with combined loss of chromosomes 1p and 19q have a more indolent clinical evolution, respond more favourably to chemotherapy and show prolonged survival compared to those with intact 1p/19q, but the basis of these clinical differences is not well understood [1–5]. In addition, up to 30% of oligodendroglial neoplasms with intact 1p/19q may respond to chemotherapy [3, 6, 7], but the genetic and biological factors associated with response are unknown and not all patients with oligodendroglial tumours derive durable benefit from therapy. Assessment of the clinical utility of biological markers and non-invasive imaging techniques is essential to advance the clinical management of these patients.
Recently dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) has been used to calculate perfusion parameters in gliomas [8, 9], guide biopsy [10], provide prognostic information [11] and demonstrate differences in the vascularity of low-grade astrocytomas compared with oligodendrogliomas [8]. It has been suggested that increased vascularity and concomitant improved drug delivery may contribute to the chemoresponsiveness of oligodendroglial tumours, especially as lipophilic drugs (procarbazine, lomustine and temozolomide), which easily cross the blood–brain barrier, are used in the treatment of oligodendrogliomas [12–14]. Currently DSC-MRI is used in the radiological evaluation of gliomas, but its role in oligodendroglial neoplasms classified by genotype and the relationship with response and outcome following chemotherapy has not so far been investigated.
The aim of this study was to determine whether DSC-MRI obtained through routine pre-therapy MR diagnostic examinations may be used to distinguish molecular subtypes of oligodendroglial tumours, and to investigate the influence of tumour blood volume on outcome following procarbazine, lomustine and vincristine (PCV) chemotherapy compared to molecular genetics.
Methods
Patient selection
Patients for investigation were selected from those participating in a prospective study of adult oligodendroglial tumours eligible for chemotherapy at the Walton Centre for Neurology and Neurosurgery/Clatterbridge Centre for Oncology between May 2000 and June 2003 [7, 15–18]. The study had full ethical approval. For inclusion in this study, patients had to have (1) given research consent, (2) a histological diagnosis of oligodendroglioma or oligoastrocytoma based on the current World Health Organisation (WHO) classification [19], (3) a known 1p/19q status, and (4) undergone DSC-MRI before or after biopsy (2 image-guided; 35 serial stereotactic), but prior to commencing therapy. All patients (with primary and recurrent disease) underwent biopsy prior to PCV. Patients with post-resection imaging were excluded.
DCS-MRI
Imaging studies were obtained using a 1.5-T Signa MR scanner (General Electric Medical Systems). The MR imaging protocol consisted of T1-weighted spin-echo (SE) sagittal sections (400/14 TR/TE), axial fluid attenuated inversion recovery (FLAIR 8802/142/2200 TR/TE/TI), axial fast spin-echo (FSE) T2-weighted imaging (4360/102 TR/TE), 2D dynamic susceptibility-weighted, SE, echoplanar imaging (EPI 1900/80 ms, bandwidth 62.5) and postcontrast T1-weighted SE (600/14 TR/TE) or FLAIR (2280/11/750 TR/TE/TI) axial imaging. The T2-weighted FLAIR, FSE and postcontrast T1-weighted images were used to define regions of T2 hyperintensity and T1 enhancement. For the dynamic EPI series, the tumour size and location, and the position of the margins were determined from the T2-weighted FLAIR or FSE images to equally define low-grade and high-grade tumours. Nine slices were selected to cover the majority of the tumour volume. Slice thickness was 7 mm, with no gap. A standard dose of 0.1 mmol/kg body weight of dimeglumine gadopentetate (Gd DTPA) was injected intravenously manually at an approximate rate of 3 ml/s. A series of T2*-weighted multislice image sets (field of view 30×30, matrix 192×128) were acquired every 2 s during the first pass of the contrast agent. Image postprocessing was performed using commercial software (Functool2, version 6.0; Sun Microsystems). Negative enhancement integral (NEI) maps were generated for each patient relative to the signal intensity in the superior sagittal sinus (Fig. 1). The axial slice with the greatest cerebral blood volume and tumour area was chosen for analysis.
Fig. 1Calculation of rCBV using DSC-MR. CBV is derived from the drop in the T2* signal after injection of a bolus of paramagnetic compound [8, 9]. Using Functool2, the radiological tumour margin was delineated on the T2* sequence axial image and a single circular ROI placed over normal brain mirrored in the contralateral hemisphere (a, b). Negative enhancement integral (NEI) colour maps were generated (c, d) relative to the signal intensity in the superior sagittal sinus using image data between dynamic scans immediately before and after the contrast transient. CBV is proportional to the area under the contrast agent concentration-time curve and was calculated for each ROI by Functool2 using algorithms to integrate ΔR2i values (where ΔR2 is the change in the reciprocal of T2*). rCBV for each tumour was calculated from signal intensity time curves for individual ROIs placed within the delineated tumour region or in the large ROI in the contralateral hemisphere (as illustrated in e and f, arrows scans adjacent to the contrast transient) as described in the Materials and methods. a, c, e Low rCBV (0.73±0.17, mean±SD) in a grade II oligoastrocytoma with intact chromosomes 1p and 19q and; b, d, f high rCBV (4.15±0.87) in a grade II oligoastrocytoma with the −1p/−19q genotype
The radiological tumour margin was delineated on the T2* sequence axial image using a nongeometric region of interest (ROI), and a single circular region was placed over normal brain mirrored in the contralateral hemisphere. Depending on tumour location, the mirrored area could include both white and grey matter. ROIs were placed according to published methods giving the lowest inter- and intraobserver variability [20] within the delineated tumour region. Briefly, five ROIs (area range 12–27 mm2) were placed over the tumour areas with the highest blood volume. Cystic and hemorrhagic regions were avoided and care was taken not to place ROIs over normal blood vessels or a biopsy tract where seen. Three independent analyses were performed by a neuroradiologist (T.S.S.) and a neurosurgeon (M.D.J.) blinded to histology and genotype, to assess inter- and intraobserver variability. For each observation, data were expressed relative to the internal reference (four ROIs placed within the delineated mirrored region of normal brain without deliberate bias towards either grey or white matter) to generate relative cerebral blood volume (rCBV) values (mean ROItumour/mean ROInormal brain) and accommodate physiological differences between patients [21]. For comparison with clinical data, the mean rCBV from these three data sets was used.
Molecular genetics
For each patient regions of tumour histology in pretherapy biopsy specimens (formalin-fixed, paraffin-embedded tissues or intraoperative diagnostic smear preparations) representative of the most aggressive tissue available and the overall pathology diagnosis were selected for laser capture microdissection to enrich the tumour component in the samples for analysis. Allelic imbalance was investigated using paired normal (microdissected uninvolved brain or DNA from blood) and tumour tissue samples and multiple simultaneous PCR amplification of a panel of microsatellite markers, capillary electrophoresis and data analysis as described previously [16–18]. The microsatellite panel included: chromosome 1 – D1S2667, D1S508, D1S214 (1p36); chromosome 19 – D19S412, D19S112, D19S596 (19q13); chromosome 17 – D17S796, D17S1176, D17S1353 (17p13); chromosome 10p – D10S89, D10S179 (10p12-10p15); chromosome 10q – D10S1687, D10s2491, D10S583, D10S587, D10S212 (10q22-10q26). Additional markers, D1S468, D1S2795 (1p36) and D19S217, D19S572 (19q13) were used in cases that lacked informativity for given loci. p53 mutations (exons 5–8) were detected by direct sequence analysis of PCR products.
PCV chemotherapy, response and clinical outcome
All patients receiving oncology therapy had evidence of advancing disease (either radiographic or clinical progression) prior to therapy. Patients with mass effect or evidence of malignancy were treated at presentation; others were followed clinically until progression. Oncology therapy was based on individual patient’s clinical status/choice. Patients fit for PCV were offered PCV chemotherapy, or radiotherapy if fertility was an issue. PCV chemotherapy was administered according to standard clinical protocols [22]: lomustine (CCNU) (110 mg/m2), procarbazine (60 mg/m2) and vincristine (1.4 mg/m2, maximum 2 mg) were given on days 1, 8–21, and 8 and 29. respectively. Cycles were repeated every 6 weeks for a maximum of six cycles. As described previously [18], a consultant neuroradiologist reviewed all available MR or CT images taken before, during and after chemotherapy, and at follow-up. The largest perpendicular diameters of the tumour were measured on axial sections of T2-weighted MR images or contrast-enhanced regions on T1-weighted MR or CT images. Response was assessed using the criteria of Macdonald et al. [23] and postcontrast T1-weighted MRI in 25enhancing tumours. In tumours showing no enhancement (n=6) or in those in which contrast enhancement was not assessable (n=1), response was based on T2-weighted images.
Information regarding current management, follow-up and outcome was collected prospectively. Progression-free survival (PFS) and overall survival (OS) were calculated from the start of PCV to progression, last seen, or death as appropriate.
Statistical analysis
Inter- and intraobserver variability was determined using Bland and Altman plots [24]. Differences in rCBV between genetic and clinical factors were assessed using Student’s t-test for numerical data and the Chi-squared or Fisher’s exact test for categorical data. Receiver operator characteristics (ROC) analysis was performed to define the optimal rCBV cut-off for predicting 1p/19q status. Kaplan-Meier survival curves were obtained and differences in OS or PFS were tested for statistical significance using the Log Rank test. Cox regression multivariate analysis of factors shown to be significant in univariate analysis was by forward stepwise entry of parameters at a significance of 0.05 for entry and 0.01 for removal. Probability (P) values less than 0.05 were considered significant.
Results
The study included 30 patients with primary tumours without previous therapy and 7 with recurrent or persistent disease following radiotherapy. Median time between previous radiotherapy and MR imaging was 6.5 years (range 1.1–14.2 years). Median age was 44 years (range 28–71 years).
Interobserver and intraobserver variability
Tumour rCBV was obtained from DSC-MRI (Fig. 1) by two independent observers. Bland and Altman plots revealed low inter- and intraobserver variation (Fig. 2). The closest observed limits of agreement were between the first observations of the neuroradiologist and neurosurgeon (mean −0.17, range −1.27 to 0.93; Fig. 2a). The widest observed limits of agreement were between the repeat observations of the neurosurgeon (mean 0.08, range −1.59 to 1.95; Fig. 2c). The greatest variation was observed in tumours with higher rCBV values.
Fig. 2Bland and Altman plots illustrating interobserver (a, b) and intraobserver (c) variation of rCBV measurements. The greatest variation is seen at the right of the plot for tumours with the higher rCBV values
Histopathology, genotype and rCBV
Oligodendroglial tumours of different histopathology subtypes and grade did not have significantly different rCBV measurements (Figure 3a,b). Loss of both 1p36 and 19q13 was seen in 6/9 grade II oligodendrogliomas, 6/14 grade II oligoastrocytomas, 4/4 grade III oligodendrogliomas, and 3/10 grade III oligoastrocytomas. Tumours with the −1p/−19q genotype had significantly higher rCBV than those with intact 1p and 19q (Fig. 3c). Similar findings were observed when primary tumours only were analysed (data not shown). ROC analysis was used to determine the specificity and sensitivity of rCBV as a potential non-invasive tool to distinguish oligodendroglial neoplasms with or without the −1p/−19q genotype. The area under the ROC curve was 0.853 (95% confidence interval: 0.778–0.927). A cut-off value of 1.59 for rCBV (sensitivity 92%, specificity 76%) best discriminated oligodendroglial tumours with and without the −1p/−19q genotype (Fig. 3c).
Fig. 3Box plots of rCBV against (a) histopathology subtype (P=0.279*), (b) histopathology grade (P=0.442*), and (c) genotype (P=0.001*) in oligodendroglial tumours. The optimal rCBV cut-off that distinguishes tumour genotype is shown by the horizontal line. *Student’s t-test
To facilitate comparison of rCBV with genotype and clinicopathological characteristics (Table 1), tumours were considered to have high rCBV if this was greater than 1.59 as determined by the ROC analysis (Fig. 4). High rCBV values were significantly more common in tumours with loss of 1p36 and 19q13 and less common in tumours with 17p13 loss and p53 mutation. Three tumours in the series had loss of chromosome 10q22–26, of which two also had loss of 10p11–15; all had high rCBV. One tumour with high rCBV had loss of 1p36 without loss of 19q13. As for numerical data, there was no significant association between high rCBV and histology subtype or grade. Contrast enhancement was weakly associated with high rCBV in the series but not in primary tumours. No associations were seen between tumour rCBV and patient age <50 years versus age ≥50 years, gender or temporal versus non-temporal location. Similar associations between rCBV, genotype and clinicopathological characteristics were obtained if only primary tumours were analysed (Table 1).
Table 1rCBV, molecular genetics and clinical characteristics in the series and primary tumours All tumoursPrimary tumoursnNumber of tumours with high rCBV P valueanNumber of tumours with high rCBV P valueaLoss of 1p36 and 19q13Yes1917 (89%)0.000*1614 (88%)0.000No184 (22%)142 (14%)Loss of 17p13Yes112 (18%)0.003*80 (0%)0.001No2619 (73%)2216 (73%)Loss of 10q 22–26 ±10p11–15Yes33 (100%)0.23811 (100%)1.0no3317 (52%)2915 (52%)p53 mutation(exon 5–8)Yes112 (18%)0.004*80 (0%)0.001No2518 (72%)2115 (71%)Patient age (years)<502513 (52%)0.491209 (45%)0.260>50128 (67%)107 (70%)Histology subtypeOligodendroglioma1310 (77%)0.091118 (73%)0.142Oligoastrocytoma2411 (46%)198 (42%)WHO histology gradeII2311 (48%)0.19199 (43%)0.118III1410 (71%)217 (78%)Contrast enhancementAbsent82 (25%)0.04682 (25%)0.092Present2819 (68%)2114 (68%)Tumour locationTemporal147 (50%)0.733136 (46%)0.713Nontemporal2314 (61%)1710 (59%)Tumour statusPrimary3016 (53%)0.674Recurrent75 (71%)Response to PCV All tumoursResponders2114 (67%)0.1421711 (65%)0.097Nonresponders114 (36%)82 (25%) Tumours with loss of 1p/19qResponders1614 (88%)1311 (85%)Nonresponders00 Tumours with intact 1p/19qResponders50 (0%)0.24540 (0%)0.515Nonresponders114 (36%)82 (25%)aProbability calculated by Fisher’s Exact test. Responders showed complete, partial or minor response and non-responders had stable or progressive disease.Fig. 4rCBV, molecular genetics and clinical characteristics. rCBV for each tumour is given as the mean±standard deviation of three independent observations. The arrow indicates tumours with rCBV above (high rCBV) and below the cut-off value of 1.59 (low rCBV). CR complete response (disappearance of all tumour, off steroids and neurologically stable or improved), PR partial response (50% or greater reduction in cross-sectional area, steroids stable or reduced, and neurologically stable or improved); MR minor response (>25 to <50% reduction in cross-sectional area, steroids stable or reduced, and neurologically stable or improved); PD progressive disease (25% or greater increase in cross-sectional area or any new tumour on CT/MR images and/or neurologically worse with steroids stable or increased); SD stable disease (all other situations); P primary tumour, R recurrent tumour
rCBV and outcome following PCV chemotherapy
Of the 37 patients, 33 had PCV chemotherapy following DSC-MRI (median 0.7 months, range 0.1–15.2 months), of whom 26 with primary tumours received PCV chemotherapy as the first oncology therapy, 6 with recurrent or persistent disease treated initially with radiotherapy received PCV as second oncology therapy, and 1 had PCV following radiotherapy and temozolomide. One patient with primary tumour had therapy switched to radiotherapy at the end of the first cycle because of reaction to procarbazine. The remainder received a median of four cycles of PCV (range one to six). Response to therapy was assessed in 32 patients (Fig. 4). Measured radiological changes and clinical factors permitted response assessment using standard criteria of Macdonald et al. [23] in all patients showing enhancement. However, in tumours showing no enhancement assessed using T2-weighted MR images, three showed radiological change in cross-sectional area of >25% to <50%, accompanied by clinical benefit; these were included in the minor response (MR) category. None of those showing enhancement had measured radiological change in this range. Response (i.e. complete, partial or minor; see Fig. 4 legend for definition) was seen in tumours with and without high rCBV. Similarly, non-responders (i.e. patients with stable or progressive disease) had either high or low tumour rCBV (Table 1, Fig. 4). Response was strongly associated with genotype; all 16 tumours with loss of 1p36 and 19q13 responded compared with 5 of 17 tumours with intact 1p36/19q13 (Chi2 P<0.001). Of the 16 responders in the 1p36/19q13 loss group, 14 had high rCBV. The five responders in the intact 1p36/19q13 group all had low rCBV and the four tumours with high rCBV failed to respond.
The relationship between rCBV, genotype, histopathology and survival following PCV in the series and in those with primary tumour is given in Table 2. The −1p/−19q genotype and low-grade pathology were associated with longer PFS and OS. Histopathology subtype was not significantly associated with outcome. Increased rCBV was only associated with short PFS and OS when adjusted for the −1p/−19q genotype. Tumours with intact 1p/19q and high rCBV (two grade III primary oligoastrocytoma, one grade II recurrent oligodendroglioma, one grade III recurrent oligoastrocytoma) were associated with shorter PFS and OS than tumours with intact 1p/19q and low rCBV or those with lp/19q loss and high rCBV (Fig. 5a,b). Similar findings were observed if primary tumour only were analysed (Fig. 5c,d). Compared with significant clinicopathological parameters, high rCBV was an independent prognostic factor for PFS when adjusted for 1p/19q status (Cox regression: PFS: hazard ratio 13.4, 95% CI 1.3–133; P=0.008) and for PFS and OS in patients with intact 1p/19q (Cox regression: PFS: hazard ratio 16.5, 95% CI 1.8–152.2; P=0.004; OS: hazard ratio 7.4, 95% CI 1.6–33.3; P=0.013).
Table 2rCBV and survival in the series and in those with primary tumours. PFS and OS were calculated from start date of PCV (n number of cases, nc not calculated, nr median survival not reached). Contrast enhancement (present vs absent), age (<50 vs ≥50 years), ECOG performance status (0–1 vs ≥2) and gender (male vs female) were not significantly related to PFS or OS nProgression-free survival (months)Overall survival (months)Median (95%CI)P valueaP valuebMedian (95%CI)P valueaP valuebAll tumoursLoss of 1p36 and 19q13Yes16>56 (nr)0.005>56 (nr)0.020No178 (0–21)>49 (nr)Histopathology subtypeOligodendroglioma11>56 (nr)0.0550.187>56 (nr)0.1130.288Oligoastrocytoma218 (9–41)50 (9–91)Histopathology gradeII19>56 (nr)0.0040.002>56 (nr)0.0070.012III144 (8–26)21 (8–34)rCBVLow14>46 (nr)0.7660.003>49 (nr)0.5850.002High1946 (nc)>56 (nr)Primary tumoursLoss of 1p36 and 19q13Yes14>56 (nr)0.037>56 (nr)0.056No12>39 (nr)49Histopathology subtypeOligodendroglioma10>56 (nr)0.0550.137All censored0.0920.209Oligoastrocytoma1646 (6–86)50 (6–101)Histopathology gradeII17>56 (nr)0.0470.009>56 (nr)0.0480.041III96 (13–37)50 (nc)rCBVLow12>46 (nr)0.6810.007>49 (nr)0.6110.003High14>56 (nr)>56 (nr)aProbability calculated by the Log-Rank test.bProbability calculated by the Log-Rank test after adjustment for 1p/19q genotype.Fig. 5rCBV and outcome following PCV chemotherapy: a, b all patients; c, d patients with primary tumour only; a, c progression-free survival; b, d overall survival.① Patients with tumours with low rCBV (<1.59) and loss of 1p36 and 19q13 (all n=2, primary n=2).② Patients with tumours with high rCBV (>1.59) and loss of 1p36 and 19q13 (all n=15, primary n=12).③ Patients with tumours with low rCBV (<1.59) with intact 1p36 and 19q13 (all n=12, primary n=10).④ Patients with tumours with high rCBV (>1.59) and intact 1p36 and 19q13 (all n=4, primary n=2)
Discussion
To our knowledge this is the first study investigating the relationship between rCBV, genotype and outcome in oligodendroglial tumours treated with PCV chemotherapy. The cohort was drawn from a larger series in an ongoing prospective study of oligodendroglial neoplasms [15–18] and reflects the range of patients given PCV chemotherapy in routine clinical practice. Tumours with rCBV >1.59 were more likely to have 1p/19q loss. PCV chemosensitivity was predicted by 1p/19q loss, but high rCBV was not necessary for response. Following chemotherapy, rCBV was significantly associated with PFS and OS only after adjustment for genotype.
DSC-MRI can be used to determine cerebral perfusion parameters such as rCBV, relative cerebral blood flow and permeability, but with the instrumentation available to us was restricted to rCBV. As in other studies [8, 20, 25, 26], placement of ROI in regions within tumour of greatest signal intensity yielded data with good inter- and intraobserver variability. The greatest variation was seen in tumours with high rCBV, reflecting heterogeneity of blood volume within these tumours. In this series, tumours with the −1p/−19q genotype were more likely to have high rCBV than those with intact 1p/19q. The ROC analysis determined an optimal cut-off (rCBV >1.59) for predicting the −1p/−19q genotype with 92% sensitivity and 76% specificity. These data suggest that in conjunction with histopathology, rCBV may be used to distinguish tumours of the oligodendroglial lineage.
MRI studies of tumour vasculature in gliomas have demonstrated that rCBV may be useful in the non-invasive determination of tumour grade and vascularity [21, 25, 27, 28]. Compared to their low-grade counterparts, high-grade tumours have elevated rCBV, associated with increased mitotic activity [29]. rCBV correlates with conventional angiographic assessment of tumour vascular density and histological measures of angiogenesis such as microvascular density and VEGF expression [26, 30–32], which reflects the histopathological finding of increasing neovascularization with grade [19]. However, most studies have investigated astrocytic gliomas or included a variety of glioma pathologies and large series of oligodendroglial neoplasms are uncommon. Recent reports suggest that rCBV values in oligodendrogliomas render DSC-MRI-based tumour grading inaccurate [28, 33], and rCBV may be similar in low- and high-grade oligodendrogliomas [33]. Indeed low-grade oligodendrogliomas have significantly elevated rCBV compared to low-grade astrocytomas [8]. In our series, rCBV did not distinguish oligodendrogliomas from oligoastrocytomas, nor was rCBV significantly different between grade II and III tumours. This lack of correlation between rCBV and grade is consistent with the increased microvessel density seen in both low- and high-grade oligodendroglial tumours [34, 35].
It has been postulated that the tumour vasculature in oligodendrogliomas contributes to their chemosensitivity [12–14]. In an anaplastic oligodendroglioma treated with PCV chemotherapy, post-therapy volume changes and normalization of rCBV were more evident in regions of the pretherapy tumour that showed high rCBV [13]. In the present study, tumours with and without the −1p/−19q genotype, and high or low rCBV showed response to chemotherapy when assessed using conventional criteria [1, 23]. These data indicate that rCBV measured through DSC-MRI is not a key factor in determining PCV chemosensitivity. These findings may reflect the limitations of DSC-MRI associated with contrast recirculation and disruption of the blood–brain barrier in tumours [8, 9]. Alternatively, chemosensitivity may be influenced more by blood flow than by volume. Further studies to measure additional parameters associated with vascular perfusion, including blood flow and permeability are essential to establish whether these factors contribute to chemosensitivity.
The −1p/−19q genotype is known to be associated with longer PFS and OS [5] but the impact of tumour vasculature on prognosis in oligodendrogliomas is confined to a few histopathological [34, 36, 37] and imaging studies [12]. Although our cohort is small, we have presented preliminary data that the prognostic significance of rCBV may differ in tumours with or without the −1p/−19q genotype. 1p/19q loss and low-grade pathology were associated with favourable PFS and OS following PCV. In other studies of astrocytic gliomas [28] or mixed low-grade gliomas [11], high rCBV predicted poor prognosis. In our series, rCBV alone was an unreliable indicator of outcome, showing prognostic significance only after stratification for genotype, suggesting different effects for the two genetic subgroups at the cut-off used to classify tumours with high rCBV. With this cut-off, tumours with high rCBV and 1p/19q loss were associated with longer survival than those with high rCBV and intact 1p/19q. Unlike astrocytic gliomas, high rCBV values do not necessarily indicate aggressive biology associated with poor outcome in oligodendroglial neoplasms with 1p/19q loss, suggestive of differences in the baseline biology of these two genetic subtypes. These preliminary data therefore indicate that further investigation of the prognostic significance of rCBV in molecular subtypes of glioma in a much larger series is warranted.
Conclusion
In conclusion, we have shown that high rCBV is more likely in oligodendroglial tumours with the −1p/−19q genotype and may be a useful aid to diagnosis; however, rCBV does not predict chemosensitivity. Lack of correlation with histopathology grade and the data suggesting that the prognostic significance of rCBV may differ in patients with or without the −1p/−19q genotype, indicates that different interpretation of rCBV data may be necessary for oligodendroglial compared with astrocytic gliomas. Further study is essential to fully evaluate the role of DSC-MRI in the non-invasive diagnosis and prediction of prognosis in gliomas with an oligodendroglial phenotype.

chemosensitivity
oligodendroglial tumour
rcbv
outcome


Determinants of potential drug–drug interaction associated dispensing in community pharmacies in the Netherlands

Objective: There are many drug–drug interactions (D–DI) of which some may cause severe adverse patient outcomes. Dispensing interacting drug combinations should be avoided when the risks are higher than the benefits. The objective of this study was to identify determinants of dispensing undesirable interacting drug combinations by community pharmacies in the Netherlands.
Statements on the impact of the article on practice
Process and structure characteristics may influence the dispensing of undesirable interacting drug combinations in community pharmacies but probably to a minor degree.
Medication surveillance in Dutch pharmacies seems to be effective.
Introduction
Drug–drug interactions (D–DIs) are responsible for many adverse patient outcomes. Different studies suggest that D–DIs may cause up to 3% of all hospital admissions [1–4]. A D–DI is defined as a pharmacokinetic or pharmacodynamic influence of drugs on each other, which may result in desired effects, in reduced efficacy and effectiveness or in increased toxicity [5]. Although many D–DIs exist, only a small part of these D–DIs is clinically relevant [6–8]. The potential benefits of drug combinations should be weighed against the seriousness of the D–DI, taking into account the availability of alternatives. Only in cases that the risks associated with the D–DI are higher than the benefits or if a better alternative is available, the D–DI should be avoided.
In the Netherlands, one of the tasks of the pharmacist is to intervene in case of D–DIs, which involve a high risk for the patient. Hereto, the pharmacist uses patient characteristics and the medication history. All prescriptions, which are submitted to the pharmacy, are screened on potential interactions with the help of medication surveillance software. These D–DIs are evaluated by the pharmacist who intervenes if necessary. This task is important but cumbersome, and requires great attention from the pharmacist. The organisational aspects, such as the tuning of the medication surveillance software and instructions of technicians, should be managed by the pharmacist in such a way that in case of D–DIs with a high risk the pharmacist intervenes. This is important for the prevention of adverse patient outcomes [9].
The objective of this study was to assess process and structure characteristics associated with the dispensing of interacting drug combinations, which carry a high risk of adverse patient outcomes.
Methods
Setting
The data for this study were retrieved from the Drug Information Project, a division of the Health Care Insurance Board. This is a database containing the reimbursement data from eight health care insurance companies in the Netherlands. The reimbursement data from January 1st, 2001 until October 31st, 2002 were analysed. Eleven potential D–DIs, that contained a high risk and could be substituted, because a good alternative was available, were selected and counted for each pharmacy in the database. These undesirable potential D–DIs were mostly interactions between chronically used drugs which cannot be interrupted and short-term use of antibiotics or antimycotics, and were selected from the Dutch guidelines for the management of D–DIs (Table 1) [10, 11]. A D–DI was counted as such, when the chronically used drug was dispensed both in the period 150 days preceding and in the period 150 days after the dispensing of antibiotics or antimycotics for short-term use in the same pharmacy. Pharmacies with less than 5,000 dispensings in the database were excluded.
Table 1Number of dispensings in the database of the individual drugs involved, the eleven potential D–DIs and the calculated ratioDrug–drug interactionNumber of dispensings drug A × 1,000 (range)Number of dispensings drug B ×  1,000 (range)Number of D–DIs counted (range)Average ratio (range)aDrug ADrug B1Erythromycin, clarithromycin, azithromycin, roxithromycinDigoxin440.8(0–2754)487.0(0–3064)3,993(0–41)1.39(0–18.52)2ItraconazoleDigoxin88.7(0–349)487.0(0–3064)245(0–7)0.45(0–21.69)3CiprofloxacinTheophylline105.4(0–769)100.9(0–756)944(0–14)6.39(0–534.38)4Miconazole oral gelAcenocoumarol, fenprocoumon44.6(0–233)608.2(5–3156)154(0–3)0.38(0–21.30)5ErythromycinCarbamazepine49.7(0–531)193.6(0–871)35(0–4)0.24(0–40.92)6Erythromycin, clarithromycin, azithromycinDisopyramide426.6(0–2754)9.4(0–151)61(0–4)–7Erythromycin, clarithromycinPimozide274.4(0–2004)57.4(0–394)70(0–15)0.46(0–46.12)8Propranolol, oxprenolol, pindololbeta2-mimetics, inhalation corticosteroids250.6(1–1075)2,546.9(27–10504)5,127(0–94)0.54(0–12.98)9Erythromycin, clarithromycinCisapride274.4(0–2004)127.5(0–821)586(0–11)1.16(0–40.45)10Itraconazole, fluconazole, ketoconazoleCisapride199.9(0–727)127.5(0–821)347(0–12)0.95(0–57.10)11Acenocoumarol, fenprocoumonAzapropazon608.2(5–3156)8.4(0–164)32(0–19)–aCalculated with Formula 1
Procedure
For each pharmacy, we calculated the dispensing-ratios for the eleven potential D–DIs with Formula 1. This formula was used because the risk of dispensing a D–DI between drug A and drug B is dependent on the number of times each drug is dispensed. The more drug A or drug B are dispensed, the higher the risk that these drugs are combined on the basis of chance alone. In case the dispensing of drug A is independent from the dispensing of drug B and the D–DI is never intervened, the ratio will on average be one. The number of times this ratio was above one was calculated, because a ratio above one might indicate that medication surveillance fails. In this calculation, there were 342 D–DIs between norfloxacin and theophylline, which were excluded in the analysis because the guidelines concerning the management of this D–DI were inconsistent. Two groups of pharmacies were selected, and the pharmacists were asked in August 2003 by the Inspectorate for Health Care (IHC) to fill in a questionnaire. The first group included pharmacies with a high risk of dispensing these D–DIs, while the second group consisted of a random sample from the remaining pharmacies. These groups were equal in size. The selection criteria are described in Fig. 1. The selection criterion for receiving a questionnaire (≥4 times a ratio >1) was chosen on pragmatic reasons to have enough power for statistical analysis with manageable numbers. A concept questionnaire was composed on basis of a literature search and interviews with experts [9]. The questions concerned process and structure characteristics of several quality aspects and those questions were selected that could discriminate between high and low quality pharmacies. Mostly questions with objective answers were included, for example about written instructions for technicians, filing of data, tuning of the software (which signals were shown and which not) and personnel. The concept questionnaire was tested in three pharmacies and some questions were amended on the basis of their comments. The final questionnaire contained 183 questions, divided into 12 subjects (Table 2). The questionnaire was accessible via the Internet. Pharmacies who had no access to the Internet received the questionnaire by post. Pharmacies who failed to fill in the questionnaire received reminders at regular intervals. A sample from both groups was visited by the IHC (Fig. 1). Also here, the selection criterion (≥5 times a ratio >1) was chosen to have enough power with manageable numbers. Thirty-seven questions from the questionnaire were selected and during the visits these questions were verified. The pharmacies were informed in advance that a selection of the pharmacies would be visited. The selected pharmacies were acquainted after completing the questionnaire. The visiting inspectors were blinded to the number of interacting drug combinations.
Fig. 1The selection of the pharmacies receiving a questionnaire and IHC visitTable 2The subjects and number of questions in the questionnaireChapterSubject (number of questions)General pharmacy dataOwnership of the pharmacy (1), cooperation with other pharmacies (1), cooperation with general practitioners (1), electronic submission of prescriptions (4)FacilitiesAlterations (2)Quality policySetting up and implementing a quality system (4), certification (2), attitude towards quality management (12)Quality measurementMeasurement of errors (2), complaints (1), patient satisfaction (2), interventions (3), and participation in mystery guest investigations (2)Receipt procedureNumber of personnel involved in dispensing a receipt (2), checks in dispensing a receipt (3)Medication surveillance—tuning softwareMedication surveillance system used (1), tuning of the system e.g. which signals are showed and which are regarded as irrelevant (55a), surveillance of pharmacy preparations (2)Medication surveillance—organisationThe way technicians are instructed to manage medication surveillance signals (5), the way this is supervised (2), number of interventions (1), use of resources (2), participation in courses (4), management of the D–DI between carbamazepine and erythromycin (5) and between Sulfamethoxazole/trimethoprim and Acenocoumarol (7)Medication surveillance—recording managementThe way the management of signals is recorded (4)Pharmacy preparationsThe way instructions for pharmacy preparations are recorded (1), the way pharmacy preparations are supervised (3), the number of pharmacy preparations (2), the policy regarding analysing pharmacy preparations (3)Personnel and workloadSubjective workload (3), absence through illness (1), number of receipts dispensed per technician (2), personnel and experience of personnel (18)Patient careInformation given to patients (6), information exchange with hospitals (4), participation in health care projects (4)Farmacotherapeutic consultation groupsParticipation in farmacotherapeutic consultation groups (3), agreements made (3)aAs the questions for the four systems (Pharmacom, Aposys, Euroned, others) differed, pharmacists had to fill in only a quarter of these questions
Formula 1:
Formula 2:
whereki,ab: number of dispensings of interacting drug combination AB in pharmacy i;ki,a: number of dispensings of drug A in pharmacy i;ki,b: number of dispensings of drug B in pharmacy i;Ni: total number of dispensed drugs known in the database in pharmacy i;Ntot: total number of dispensed drugs known in the database in all pharmacies
Statistical analysis
For each pharmacy, dispensing-ratios for the D–DIs, comparable to the standardised mortality ratio, were calculated using Formula 2. With this formula, we standardise for the total number of dispensings per pharmacy in the database. In case all pharmacies dispense the D–DIs in equal numbers, the ratio will be one for all pharmacies, and therefore the ratios have a better comparability. Pharmacies which have only a small number of dispensings in the database, will have extremely high numbers in case they dispense one or a small number of D–DIs. Therefore, the results were equalized with Bayesian statistics to prevent extreme ups and downs due to low numbers of dispensings [12]. The pharmacies were divided into two sets. One set was used for the analyses and contained two-third of the pharmacies, the other set was used for the validation of the results obtained in the analyses. In the univariate analysis, correlations were searched between the answers in the questionnaire and these ratios. Correlations are only given if in both sets a correlation was found (p < 0.01). In the multivariate analysis, models were composed using the analysis set, predicting the dispensing of the interacting drug combinations. The models were validated using the validation set. The number of questions was too large for the multivariate analysis, and only a limited number of questions were selected. From every chapter, those questions were selected that correlated with the other questions and that could discriminate between pharmacies.
Results
The database contained a total of 100,295,311 dispensings in the selected study period. One thousand one hundred and forty-two pharmacies were recorded in the database with 5,000 or more dispensings. The number of dispensings per pharmacy varied from 5,019 to 264,631. Because pharmacies receive reimbursements from several health care insurance companies and because not all health care insurance companies were included in the database, these numbers do not correspond with the total number of dispensings per pharmacy. The eleven potential D–DIs were dispensed 11,594 times. In 5%, more than one pharmacy was involved. As these D–DIs could not be assigned to a single pharmacy, they were excluded from further analyses. The number of dispensings and D–DIs are shown in Table 1. Disopyramide (D–DI number 6) and azapropazon (D–DI number 11) were not dispensed by 44% and 46% of the pharmacies, respectively. Therefore, a ratio could not be calculated for these pharmacies and these D–DIs were excluded from the analyses.
The number of times a ratio above one was found was calculated (Table 1) and pharmacies were selected as shown in Fig. 1. Two hundred and sixty-eight pharmacies were selected to receive a questionnaire and 74 pharmacies were selected for a visit by the IHC. For several reasons, such as recent visitations and duplications in the database, 12 pharmacies were excluded. Eventually, 256 pharmacies received a questionnaire and 62 pharmacies were selected for a visit. Two hundred and forty-six questionnaires were filled in (response rate 96.1%) and 58 (93.5%) pharmacies were visited after the questionnaire was completed. The judgements during the visits by the IHC were compared with the answers by the pharmacists. In 33 of the 37 verified questions, the IHCs judgement matched in more than 90% the answer of the pharmacist. Except four questions, the judgement by the IHC was equally more positive and more negative than the answers by the pharmacist.
In the univariate analysis, all combinations between the questions and D–DIs were searched for significant correlations. Two correlations were found with D–DI number 1 between macrolide antibiotics and digoxin (Table 3). Pharmacies, which are part of a health care centre dispensed this interacting drug combination more often than other pharmacies. A correlation with the type of medication surveillance system was also found. Pharmacies using the Euroned system dispensed this interacting drug combination more often, while pharmacies using the Pharmacom system dispensed this interacting drug combination less often.
Table 3Significant univariate correlations between the questionnaire and the number of dispensings of the D–DIs between macrolide antibiotics and digoxin (number 1)QuestionCorrelationSignificanceIs the pharmacy part of a health care centre? (1 yes, 2 no) (yes n = 18, no n = 228)−0.1650.009Which medication surveillance system is used in the pharmacy?Pharmacom (1 yes, 0 other) (n = 81)−0.2610.000Aposys (1 yes, 0 other) (n = 62)0.0880.170Euroned (1 yes, 0 other) (n = 89)0.1970.002
For the multivariate analysis, 32 variables were selected, representative of the whole range of questions. These variables were used in the analysis-set to compose models. The adjusted explained variance ranged from 2.6% to 28.9% (Table 4). The model explaining the D–DI between macrolide antibiotics and digoxin had by far the highest adjusted explained variance. The models were validated in the validation-set, calculating the unexplained variance (Table 4). The six variables in this model explaining the D–DI between macrolide antibiotics and digoxin are shown in Table 5.
Table 4Predictability of the models composed in the multivariate analysisD–DIAdjusted explained variance (R2) (analysis set)Unexplained variancea (validation set)128.90.61212.8−0.22317.331.547.0−0.18514.4−0.4176.56.4816.10.68914.0−0.43102.60.90aAn unexplained variance of zero means that the predictability found in the validation set equals the predictability in the analysis-set. The higher the unexplained variance, the worse the predictability in the validation-set compared to the analysis-setTable 5The questions in the multivariate model predicting the dispensing of the D–DI between macrolide antibiotics and digoxin (number 1)Variable:Answer (coding)Direction coefficientConstant3.3679Is the pharmacy part of a health care centre? (yes n = 18, no n = 228)Yes (0) versus no (1)−2.2749Co-trimoxazole—acenocoumarol: no appointments were made with the GPs. The drug will be dispensed.Option 1 ‘with all GPs’ (1) versus other option (0) (n = 11)ReferenceEight options of choice option 1 ‘with all GPs’ and optionOption 2 (1) versus other option (0) (n = 10)1.0308Eight ‘with no GPs’Option 3 (1) versus other option (0) (n = 4)0.3788Option 4 (1) versus other option (0) (n = 4)−0.4542Option 5 (1) versus other option (0) (n = 3)0.9026Option 6 (1) versus other option (0) (n = 2)−0.5100Option 7 (1) versus other option (0) (n = 4)−0.1912Option 8 ‘with no GPs’ (1) versus other option (1) (n = 202)0.0886Are separate signal texts in the medication surveillance program adjusted to the situation in the pharmacy? (yes n = 72, no n = 165)Yes (0) versus no (1)0.1793Is the management of signals traceably recorded on the receipt? (yes n = 211, no n = 35)Yes (0) on the receipt, no not on the receipt (1)0.2691The supervision on management of signals takes place on the basis of signal lists (yes n = 158, no n = 86)Yes (0) on the basis of signal lists, no (1) not on the basis of signal lists0.0723How many receipts are dispensed per year divided by the number of fte technicians<10–4
Discussion
In this study, we investigated determinants for the dispensing of 11 undesirable interacting drug combinations. In general, our results are in line with the expectation that the medication surveillance system plays an important role in medication surveillance. Although the 11 potential D–DIs were counted 11,594 times which suggests that a considerable number of patients is exposed to potential and avoidable adverse patient outcomes, these results should be judged against a background of approximately 100 million dispensings. It is possible that in these cases due to particular circumstances any other option, such as substituting or not dispensing one of the drugs, is a less favourable choice than dispensing the D–DI. In 5% of the total number of D–DIs more than one pharmacy was involved, indicating the importance of communication. For the D–DI between macrolide antibiotics and digoxin, two determinants were found. Although the type of medication surveillance system was a determinant, this does not mean that the differences are determined by the quality of the system itself because they may also correlate with the attitude of the pharmacists using the systems. The three medications surveillance systems differ in the extent to which communication with other healthcare providers is possible and developments were made in recent years. The Pharmacom system has the most advanced communication possibilities and compared to the other systems, new developments to the Euroned system were modest. Unexpectedly, pharmacies part of a health care centre dispensed this D–DI more often than other pharmacies. In health care centres, the communication lines between pharmacists and general practitioners are much shorter, suggesting that intervening undesirable D–DIs will be easier. Possibly, pharmacies which are part of a health care centre oppose the opinions from the general practitioners less often, to avoid harming the cooperation within the health care centre but, of course, there may be several other reasons.
For the other eight assessed D–DIs no determinants were found in the univariate analysis, neither did the models in the multivariate analysis have a good predictability. A possible explanation is that the quality of medication surveillance in community pharmacies in the Netherlands is high. Therefore, the number of pharmacies dispensing high-risk D–DIs seems to be small.
Our study has some potential limitations. First, because we used strict inclusion criteria to prevent false-positive results, it is likely that the number of dispensings of undesirable interacting drug combinations in this study is an underestimation and it is possible that important determinants were not recognized or difficult to assess. In the univariate analyses, only those questions are given which had a significant (p < 0.01) correlation in two independent sets. Although we included 183 questions and nine D–DIs in the univariate analysis, the possibility of including a significant correlation by chance was small (on average 0.16 question). Second, the reimbursement data from eight health care insurance companies were used. In the Netherlands, these companies work mostly regionally. It is nevertheless not to be expected that the determinants of dispensing interacting drugs differ per region or that pharmacies differ in their management of D–DIs between patients of different health care insurance companies. Third, from all potential D–DIs, only 11 (but highly clinically relevant ones) were selected for this study. According to the Dutch guidelines, for all 11 combinations the dispensing of an alternative was strongly advised as a good alternative was available. Nevertheless, it is possible that these dispensings were not an error because any other option was not possible. For example, when a patient is hypersensitive to the alternative drug recommended in the guidelines or when the alternative drug is not effective. In these cases, the benefit of both drug therapies should be weighed against the potential risks of the D–DI. The potential risks can partly be avoided by taking appropriate measures such as monitoring of drug levels. In this study, we could not retrieve why the pharmacist had dispensed the interacting drug combination, and whether the dispensing was erroneous or not.
Fourth, the questionnaire was composed on the basis of a literature search and interviews with experts. It is possible that not all characteristics correlating with the dispensing of undesirable interacting drug combinations were disclosed, such as differences in population characteristics between pharmacies. For example, pharmacies with an elderly population using more drugs simultaneously have a higher risk of dispensing interacting drug combinations than pharmacies with a younger population. Also, it is possible that in areas with many general practitioners who use a medication surveillance system for prescribing, the background chance of a D–DI is much smaller. Fifth, it is possible that the differences between pharmacies were too small compared with the power of this study to distinguish determinants.
All associations found in this study were directly related to the management of signals. In our questionnaire, we also included other topics, such as pharmacy preparations and patient care. Future research should focus on the management of a larger variety of signals than the ones in our study and on how D–DI associated dispensing could be further reduced.
Conclusion
In conclusion, both medication surveillance systems and being part of a health care centre may play an important role in the management of D–DIs and the avoidance of adverse patient outcomes. Pharmacies in a healthcare centre dispensed D–DIs more often. For most D–DIs, no determinants were found possibly indicating that the quality of medication surveillance in the Netherlands is high.

drug–drug interaction
pharmacy
netherlands
drug interaction
practice management


Control and possible applications of a novel carrot-spoilage basidiomycete, Fibulorhizoctonia psychrophila

A novel cold-tolerant fungus, Fibulorhizoctonia psychrophila, was isolated from a refrigerated carrot storage facility and identified as an anamorph of Athelia, often classified in Rhizoctonia s.l. Growth of this fungus was observed between 0 and 20°C with an optimum at 9–12°C, while incubation of mycelium grown at 15–32°C resulted in absence of growth even after the fungus was transferred back to 15°C. Growth was inhibited in the presence of the antifungals sorbic acid or natamycin, in particular when the fungus was incubated at 18°C. F. psychrophila produces polysaccharide degrading enzymes that, when compared to enzymes from the ascomycete fungus Aspergillus niger, retain a larger proportion of their activity at lower temperatures. This indicates that F. psychrophila could be used as a source for novel industrial enzymes that are active at 4–15°C.
Introduction
Fungi occupy every natural environment on earth as well as man-made indoor environments. In fact, some fungi have ecological requirements and amplitudes that are ideally suited to some artificial (human made) environments. Some of these fungi create large problems with respect to human health (e.g. Aspergillus, Candida) or to food quality (e.g. Penicillium). Among standard methodologies to prevent food spoilage are the use of preservatives and refrigeration of food. The latter method, however, is not effective when cold-tolerant spoilage fungi are present. Cold-tolerant fungi are commonly found in nature and comprise species of different branches of the fungal kingdom, such as Geomyces, Leucosporidium (Panikov and Sivoza 2007), Cystofilobasidium and Mrakia (Nagakawa et al. 2004).
Cell walls form the majority of the plant biomass and consist mainly of polysaccharides. As fungi are not able to import polymeric compounds into their cells, they rely on extracellular enzyme systems for the degradation of polysaccharides into monosaccharides and short oligosaccharides. Hydrolytic enzymes acting on polysaccharides are commonly produced by fungi. These enzymes have many applications in the food and feed, paper and pulp, textile, and pharmaceutical industries and have therefore been the subject of many studies (de Vries and Visser 2001; de Vries 2003). Most of these studies and applications involve enzymes of saprophytic ascomycete fungi (mainly species from Aspergillus and Trichoderma) that have optimal growth temperatures between 30 and 37°C. Consequently, most of these enzymes have optimum activities between 30 and 50°C and their activity rapidly decrease at lower temperatures (de Vries and Visser 2001). Efficient use of these enzymes in industrial applications is therefore often only possible by incubation above 30°C. This not only increases production costs due to the energy required to reach this temperature, but can also cause spoilage problems, especially with respect to food and feed applications.
One of the most commonly known cold-tolerant spoilage fungi is the basidiomycete Rhizoctonia carotae (Adams and Kropp 1996; Jensen 1969; Jones and Aldwinckle 1990; Punja 1987), which is found in storage facilities of several vegetables such as carrot. Although the optimum growth-temperature of this fungus is between 15 and 20°C (Punja 1987), this fungus causes crater rot characterized by sunken lesions and abundant mycelial growth on carrots stored at temperatures between 1 and 4°C. The anamorph genus Fibulorhizoctonia (Adams and Kropp 1996) (as Fibularhizoctonia) was proposed to accommodate Rhizoctonia carotae and R. centrifuga Lév (Rader 1948). The teleomorphs of these species belong to Athelia Pers., while those of the Rhizoctonia solani-complex belong to Thanatephorus Donk, currently placed in a different order (Hibbett et al. 2007). The teleomorph of F. carotae was identified as Athelia arachnoidea (Berk.) Jülich (Adams and Kropp 1996). However, there is some controversion considering the scope of this species, as the name has been used for litter decomposing specimens and lichen parasites. Arvidsson (1976) concluded that the use of the name A. arachnoidea should be confined to the lichen parasite, while the material of Adams and Kropp (1996) contained only litter decomposers.
Other basidiomycete species reported from cold storage are: Corticium centrifugum (Bielenin 1986; Stalpers and Loerakker 1984; Weresub and Illman 1980), from stored apples and pears, causing fisheye rot. Although the name C. centrifugum is connected with Athelia, the species concerned differs by having constant clamps at the septa, narrower hyphae and no sclerotia, and the production of a basidiome in culture, which is now known as Butlerelfia eustacei (Weresub and Illman 1980). This species is known from Europe and North America.
In this paper we describe a new carrot-spoilage fungus, Fibulorhizoctonia psychrophila, which is a predominant species detected in refrigerated storage facilities for carrots in The Netherlands. We have analysed the cold-tolerance of this fungus and its sensitivity to commonly used fungicides. In addition, we have analysed F. psychrophila for production of plant polysaccharide degrading enzymes to assess its potential as a source of cold-active enzymes.
Materials and methods
Strains and growth conditions
F. psychrophila CBS 109695 (IMI 395943) was isolated as a mycelial sample by H.A.B. Wösten from a wooden crate containing Daucus carota in a refrigerated storage facility (4°C) in Bant, The Netherlands in 2002. Athelia arachnoidea (Berkeley) Jülich CBS 418.72 was isolated from fallen leaf-litter of Populus sp. in The Netherlands. The type strain of Fibulorhizoctonia carotae (Rader) G.C. Adams & Kropp CBS 464.48 and Aspergillus niger N402 were described previously (Adams and Kropp 1996; Bos et al. 1988).
F. psychrophila CBS 109695 and A. niger N402 were routinely propagated on malt extract agar and minimal medium (de Vries et al. 2004), respectively. For growth of F. psychrophila on carrot, potato and onion, these vegetables were ground using a coffee grinder. The vegetable pulp was used at a concentration of 10% in 1.5% agar plates in water. Liquid cultures of F. psychrophila and A. niger were performed in Schizophyllum commune minimal medium (Dons et al. 1979) and Aspergillus minimal medium (de Vries et al. 2004), respectively, using 1% of a crude arabinoxylan preparation obtained from wheat after extraction of starch and proteins as the substrate. Plate cultures to determine the influence of several fungicides on growth of F. psychrophila were performed on malt extract agar plates. Comparison of the optimal growth temperature for F. psychrophila, Athelia arachnoidea and F. carotae was performed on malt extract agar and cherry decoction agar at temperatures of 0–27°C with intervals of 3°C.
Powdered wood was obtained by grinding wood shavings of a storage crate in a coffee grinder until a fine powder was obtained. Of this powder, 1 g was added to 1.5 g agar and 100 ml water and autoclaved to prepare solid media. The crude arabinoxylan preparation was a gift from Latenstein (Nijmegen, The Netherlands) and is in fact a waste stream of a protein and starch extraction process from wheat.
Molecular biology methods
Genomic DNA of CBS 109695 was extracted using the FastDNA kit (Bio 101 Systems, Q-Biogene). The 5.8S gene and flanking ITS1 and ITS2 were amplified using the primers ITS1 and ITS4 (White et al. 1990) and the sequence was deposited at genbank (Acc. Nr. EF492880). The sequence was compared with ITS sequences from F. carotae (U85789) and A. arachnoidea (U85791).
Enzyme assays
F. psychrophila and Aspergillus niger were grown at 15 and 30°C, respectively in liquid medium containing a crude arabinoxylan preparation. Culture filtrate was harvested over a nylon filter after 2 days of growth and analysed for enzyme activities at 4, 15 and 30°C. Enzyme activity was determined using p-nitrophenyl-β-d-galactopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-α-d-galactopyranoside, and p-nitrophenyl-β-d-xylopyranoside (Sigma) as a substrate. A mixture was made consisting of 10 μl culture filtrate, 50 μl 50 mM sodium acetate (pH 5.0) and 30 μl sterile MiliQ water. The reactions were performed in triplicate and were started by the addition of 10 μl of 0.1% stock of the p-nitrophenyl-linked substrate and incubated for 2 h at 25°C, unless stated otherwise. The reactions were stopped by the addition of 100 μl 0.25 M Na2CO3 and measured at 405 nm in a micro plate reader (model 550, Bio-RAD). The amount of free p-nitrophenol was calculated using a calibration curve.
Results and discussion
Taxonomy
Fibulorhizoctonia psychrophila Stalpers & de Vries spec. nov. (Fig. 1).
Fig. 1Colony morphology of Fibulorhizoctonia carotae (464.84), Athelia arachnoidea (418.72) and Fibulorhizoctonia psychrophila (109659) after 2 weeks. (a) 0°C; (b) 6°C; (c) 18°C
Mycelium ad 9°C cotoneum, albidum, ad 18°C cremeum, avellaneum vel brunneum, velutinum vel crustosum. Liquor exsudatus brunneus. Sclerotia presentia vel absentia, irregularia, in statu maturitate subfusca a liquore exsiccata. Hyphae hyalinae vel subfuscae, (2.5–)3–6 μm. Septa fibulatae vel afibulatae. Cellae sclerotiorum doliformes, tenui-tunicatae vel crasse-tunicatae, hyalinae vel fuscae. Fungus psychrophilus, ad temperaturas −3° ad 21°C crescens.
Typus: CBS 109695
Colonies at −3 to 9°C are cottony, rather high and reaching the lid of the Petri dish, white. Margin raised, rather dense. At increasing temperatures the mycelium grows less high and becomes nearly velvety at 18°C and nearly crustose at 21°C. At 12° the centre of the colony is cream-coloured, at 15°C becoming Light Cinnamon Drab to Avellaneous (Munsell 7.5YR6/2, 10YR6/2, 10 YR6/3). At these temperatures exudate droplets are not present. At 18°C the colony is low, nearly velvety, Ochraceous Tawny (7.5YR5/8) caused by exudate drops and at 21°C there is hardly any growth; the mycelium is crustose and Cinnamon Brown to Prouts Brown (5YR4/3, 5YR4/4), and finally Mummy Brown. Sclerotia are generally not produced within 6 weeks, but have occurred in a tube; they start as white semiglobose pustules, generally up to 1 mm diameter, aggregated, often fusing, and producing a brown exudate, which dries in, leaving a dark brown sclerotium, up to 5 mm diam, finally Mummy Brown.
Marginal hyphae at −3 to 3°C are irregular, rather long-celled, with swellings and granular contents, thin-walled, (2.5–)3–6 μm wide, swellings up to 12 μm wide. Clamps present, not abundant. Branching at various angles, sometimes a constriction is present at the base of the side branch. At 6°C and above the hyphae are regular, thin- to firm-walled, 2.5–6(–7.5) μm wide, contents hyaline to pale brownish, with abundant clamps, especially at the wider hyphae, but the narrower hyphae (2.5–3.4–4 μm) generally have clampless septa. Cell wall hyaline, but irregular brownish encrustations present at some hyphae, probably dried exudate.
Aerial mycelium regular, thin- to firm-walled, (2.5–)3.2–6.5 μm wide, with hyaline to brownish contents. Most septa with clamps; clamps regular, but sometimes of the medallion type.
Sclerotia rarely produced, uniform, not forming a distinct cortex, consisting of thin- to thick-walled hyphae, usually consisting of slightly elongated swollen cells with granular contents, 4–10 μm wide, not unlike the (thin-walled) advancing hyphae at lower temperatures.Cardinal temperatures: minimum below −3°C, optimum range between 9 and 12°C, maximum 20°C.Holotype: CBS 109695 (IMI 395943), also preserved dried in herb. CBS.Mycobank number: MB501325.The sterile, sclerotium producing mycelia are traditionally classified in the artificial genus Rhizoctonia, which is currently restricted to basidiomycetous fungi and comprises anamorphs of various, not closely related genera such as Tulasnella, the Ceratobasidium–Thanatephorus complex and Athelia (Stalpers and Anderson 1996). These groups have been elevated to the genus level as Epulorhiza, Rhizoctonia s.str. and Fibulorhizoctonia. Although teleomorphs have not been observed in F. carotae and F. psychrophila, morphological, physiological and molecular characters indicate that F. psychrophila, F. carotae and Athelia arachnoidea are closely related and belong to Athelia, a corticioid genus, currently classified in the Atheliales (Rader 1948).
F. carotae has been described from cold stored carrots. It differs from F. psychrophila in growing between 18 and 27°C (Fig. 2), abundant production of sclerotia, and colour of the mat above 15°C. A. arachnoidea has been described from both lichens and leaf litter. It differs from F. psychrophila in its growth at higher temperatures (Fig. 2), less abundant aerial mycelium, and pale colour at higher temperatures. ITS sequencing of F. psychrophila revealed 95% identity to the ITS sequence of A. arachnoidea and A. carotae (data not shown), indicating that F. psychrophila is very closely related to these two species.
Fig. 2Growth comparison at different temperatures of Fibulorhizoctonia psychrophila, Fibulorhizoctonia carotae and Athelia arachnoidea. The means of duplicate values are represented
Both morphological and molecular characters indicate without doubt, that F. psychrophila, F. carotae and Athelia arachnoidea are closely related.
Temperature-tolerance and growth of F. psychrophila
F. psychrophila was inoculated on malt extract agar plates and incubated between 0 and 27°C. Growth was monitored by measuring the colony diameter. Optimal growth occurred at 9–12°C (Fig. 2) and growth was absent above 20°C. When plates incubated for 3 days at 20 and 25°C were placed back at 15°C the fungus re-initiated growth, but incubation for 3 days at 32°C prevented growth of F. psychrophila after the plates were transferred to 15°C (data not shown). Optimal growth of F. carotae and A. arachnoidea was at between 18 and 21°C (Fig. 2). The strong decrease in growth of F. psychrophila when the incubation temperature is raised above 15°C, suggests an even stronger adaptation of this fungus to cold biotopes.
Growth of F. psychrophila was tested on three vegetable crops that are commonly stored under refrigerated conditions (carrot, potato and onion) in comparison to agar plates without carbon source. Water agar alone already permits growth of F. psychrophila, but growth is significantly improved in the presence of carrot (data not shown). The presence of potato only resulted in a small increase in growth compared to water agar, but onion reduced growth of F. psychrophila. These data demonstrate a preference of F. psychrophila for carrot as a substrate. The reduced growth on onion is likely caused by the high levels of phenolic compounds in onions.
Inhibition of growth of F. psychrophila using antifungals
Sorbic acid and natamycin are compounds commonly used for the inhibition of growth of spoilage fungi and the effectiveness of these compounds was tested on F. psychrophila on malt- and water agar plates with powdered wood. F. psychrophila is believed to survive on the crates used for the storage of carrots and therefore causes repeated spoilage when these crates are re-used. The presence of 250 mM sorbic acid resulted in a significant reduction in growth on malt extract agar, but not on powdered wood at 4°C (Fig. 3a). However, higher levels of sorbic acid (625 and 1,250 mM) prevented growth of F. psychrophila on both substrates. In light of the high concentrations of sorbic acid required to inhibit growth, the expected effectiveness of this compound in preventing spoilage by F. psychrophila is limited.
Fig. 3Influence of antifungals on the growth of Fibulorhizoctonia psychrophila grown on malt extract agar (MA) or powdered wood from storages crates (HW). (a) sorbic acid, 18°C; (b) natamycin, 4°C; (c) natamycin, 18°C
Natamycin was more effective in preventing growth of F. psychrophila, resulting in a significant decrease in growth at 2 g/l at 4°C. Increasing levels of natamycin reduced growth of the fungus at 4°C similarly on both malt extract agar and powdered wood, although a significant difference was observed at 50 and 100 g/l (Fig. 3b). However, at 100 g/l natamycin, some residual growth could still be observed for powdered wood. A stronger inhibition with natamycin was observed when the incubations occurred at 18°C instead of 4°C, resulting in nearly complete inhibition of growth on both media at 0.5 g/l natamycin (Fig. 3c). The results described here indicate that spraying wooden crates with natamycin and incubating them at 18°C or higher likely reduces spoilage F. psychrophila.
F. psychrophila produces cold-active hydrolytic enzymes during growth on polysaccharides
Both carrots and powdered wood consist largely of polysaccharides. As these are the main carbon sources for F. psychrophila during growth in the storage facilities, it can be expected that the fungus produces polysaccharide degrading enzymes that are active at low temperatures. To study whether these enzymes have in fact a higher relative activity at low temperatures than those currently used in industrial applications, we compared enzyme activities from F. psychrophila to those from Aspergillus niger. A. niger is one of the most commonly used fungi for the production of industrial enzyme preparations and produces a wide range of polysaccharide degrading enzymes (de Vries and Visser 2001). However, these enzymes are mainly active at higher temperatures.
For A. niger, enzyme activities dropped to 30–50% when incubation at 15°C was compared to 30°C (Fig. 4a). At 4°C, only 10–20% of the 30°C-activity was observed. For F. psychrophila only a 5–35% drop was detected when comparing 30°C to 15°C and 30–60% of the activity at 30°C was still observed at 4°C (Fig. 4b). The strongest difference was observed for α-galactosidase and β-glucosidase, where more than 90% of the activity was still detected at 15°C for F. psychrophila, while for A. niger only 35–45% of the activity was detected at this temperature.
Fig. 4Relative hydrolase activities in culture filtrate of Fibulorhizoctonia psychrophila and Aspergillus at different temperatures. (a) A. niger culture filtrate; (b) F. psychrophila culture filtrate. AGL = α-galactosidase, LAC = β-galactosidase, BGL = β-glucosidase, BXL = β-xylosidase. Activity at 30° is set at 100%
F. psychrophila and other cold-tolerant fungi are potential sources for enzymes with high activity at low temperatures. This idea is supported by studies with cold-tolerant yeast and fungi, which were shown to produce cold-active pectinases (Nagakawa et al. 2004, 2005a, b), although in these studies a direct comparison with industrially used enzymes was not made. Cold-active enzymes can be important for many applications, for example in detergents or for the removal of lactose from milk for lactose-intolerant people.

carrot-spoilage
fibulorhizoctonia psychrophila
cold-tolerant fungus
cold-active enzymes