**3.1 Low molecular weight cut off**

In a landmark Swedish study in 2003 Roslin et al studied the baseline concentration of glucose and its metabolites, glycerol and glutamate in patients with high grade glioma

Clinical Microdialysis in Glioma 153

with traumatic brain injury was included as a non-tumour control. All but one of the patients with cerebral tumours underwent surgical resection with the first microdialysis catheter placed at the tumour resection margin (T) and the second inserted at least 20mm away in macroscopically unaffected brain around tumour (BAT). The remaining one patient had an image-guided biopsy of their tumour with stereotactic insertion of a catheter into the tumour margin (T). Microdialysates were first assessed for small molecules using the CMA 600 or ISCUS analyser. Tumour microdialysates were found to have a significantly lower glucose, higher lactate/pyruvate (L/P) ratio, higher glycerol and higher glutamate compared to the brain around tumour. These findings suggest that the tumour margin of HGGs is particularly metabolically active and are consistent with previously published invitro and animal studies, but differ from the previous clinical microdialysis study by Roslin et al. There are a several reasons that may account for the discrepancy between these studies. First, the small number of cases in both studies necessitates cautious interpretation of their findings as variation in patients, their pathology and tumour heterogeneity may all have influenced the concentration of glucose and its metabolites, glycerol and glutamate. Second, Roslin et al introduced catheters following biopsy using a closed stereotactic technique while Marcus et al introduced catheters after tumour resection using an open technique. Trauma artefact may therefore have influenced findings (though in the Marcus et al study measurements were taken at least 4 hours post-operatively to try and reduce this effect). Third, while Roslin et al placed the tumour catheters in the tumour centre, Marcus et al placed tumour catheters at the tumour resection margin or tumour periphery. It is possible that the core of the tumour, which is often necrotic, may be less metabolically active

In the same study Marcus et al analysed all the remaining microdialysate samples for macromolecules using a sandwich ELISA like procedure. There was great variability in the dialysate concentrations of the various growth factors (TGF-alpha, VEGF, EGF), cytokines (IL-1a, IL -1b, IL-1ra, IL-6, IL-8) and matrix metalloproteases and their tissue inhibitors (MMP2, MMP9, TIMP1, TIMP9). Nevertheless, microdialysates were found to have significantly raised MMP2/TIMP1 and IL-8 in T compared to BAT samples suggesting an

In addition to using microdialysis to evaluate the baseline concentration of molecules involved glioma pathogenesis, several studies have also made use of the technique to

Microdialysis may be used to evaluate both chemotherapeutic pharmacokinetics and pharmacodynamics. Interestingly, the earliest example of clinical microdialysis in patients with brain tumours to investigate a drug's pharmacology focused not on chemotherapy but on the antimicrobial rifampicin. Mindermann et al recruited 5 patients with HGG and 3 patients with LGG (Mindermann 1999). All patients received a single pre-operative dose of 600mg rifampicin 3 hours before skin incision. Patients then underwent craniotomy and tumour resection with a single microdialysis catheter placed distantly from the resection margin in grossly unaffected brain around tumour (BAT). A LWCO catheter was infused

environment favouring invasion and angiogenesis respectively.

investigate the response to treatment with chemo- and radiotherapy.

than the brain-tumour interface.

**4. In vivo assessment of therapies** 

**4.1 Chemotherapy** 

(HGG) (Roslin et al. 2003). The group performed an in-vitro recovery experiment, which confirmed relative recoveries of greater than 90% of the substrates of interest. Fifteen patients with HGG undergoing brain biopsy were recruited and two LWCO catheters were placed stereotactically: one within the tumour (T), and one 10mm outside the contrast enhancing region in the peritumour region (PT). Surprisingly, the only significant difference between dialysates obtained from T and PT was lactate, which was more concentrated in T than PT (p<0.05). This is in contrast to in-vitro studies (Klegeris, Walker and McGeer 1997), animal studies (Behrens et al. 2000), and subsequent clinical microdialysis studies (Marcus et al.), all of which demonstrate an increased concentration of glutamate and other metabolites within tumour cell lines and tissue respectively. The possible reasons for this incongruity are discussed below (see Section 3.2)

Investigators in Italy also used LWCO microdialysis catheters to establish the baseline concentration of other small molecules including amino acids, adenosine, and choline in 21 patients with HGG (Melani et al. 2003, Bianchi et al. 2004). An in-vitro recovery experiment was carried out and demonstrated the relative recovery of adenosine estimate to be 43.4 ± 5.1% (relative recovery was not calculated for amino acids and choline). Unlike the Swedish study patients underwent tumour resection and three microdialysis catheters were placed using an open technique: one into the tumour resection margin (T), one into peritumoural tissue 10mm away from the resection margin (PT), and one into grossly normal brain around the tumour at least 20mm away from the resection margin (BAT). Samples were analysed using various methods including HLPC. The group found that concentration of adenosine and glutamate were significantly reduced in T compared with BAT (p<0.05), the concentration of aspartate was unchanged, and the concentration of the remaining amino acids and choline were all significantly increased in T compared with BAT (p<0.01). Interestingly epilepsy, which occurs in approximately a third of patients with brain tumours (Villemure and de Tribolet 1996), was found to be an important confounding variable when the concentrations of aspartate, glutmate and GABA were considered.

#### **3.2 High molecular weight cut off**

Flannery et al were the first group to take advantage of HWCO catheters to assess the cysteine protease Cathepsin S (CatS) in gliomagenesis (Flannery et al. 2007). In total 11 patients with suspected HGG were recruited. Of these 11 patients, one was subsequently found to be a low-grade glioma (LGG), 2 were cerebral metastases, and the remaining 8 cases confirmed HGGs. A further patient with suspected hydrocephalus that was undergoing intracranial pressure monitoring was also included as a control. All patients underwent tumour resection with insertion of a single microdialysis catheter at the tumour resection margin (T). Analysis of CatS was by activity and ELISA concentration assays. Unfortunately, the absence of paired catheter data makes interpretation of the study's findings difficult but there was no significant relationship between CatS concentration and function, and the grade of brain tumours investigated.

A more recent study utilising HWCO catheters in patients undergoing surgery for intrinsic brain tumours set out to first to repeat earlier measurements of glucose and its metabolites, glycerol and glutamate, and second to assess the concentration of macromolecules such as growth factors, cytokines and other proteins involved in the pathogenesis of HGGs (Marcus et al.). Eight patients with suspected HGG were recruited. Of these 8 patients, one was found to have a lymphoma, and the remaining 7 cases confirmed HGGs. A further patient

(HGG) (Roslin et al. 2003). The group performed an in-vitro recovery experiment, which confirmed relative recoveries of greater than 90% of the substrates of interest. Fifteen patients with HGG undergoing brain biopsy were recruited and two LWCO catheters were placed stereotactically: one within the tumour (T), and one 10mm outside the contrast enhancing region in the peritumour region (PT). Surprisingly, the only significant difference between dialysates obtained from T and PT was lactate, which was more concentrated in T than PT (p<0.05). This is in contrast to in-vitro studies (Klegeris, Walker and McGeer 1997), animal studies (Behrens et al. 2000), and subsequent clinical microdialysis studies (Marcus et al.), all of which demonstrate an increased concentration of glutamate and other metabolites within tumour cell lines and tissue respectively. The possible reasons for this incongruity

Investigators in Italy also used LWCO microdialysis catheters to establish the baseline concentration of other small molecules including amino acids, adenosine, and choline in 21 patients with HGG (Melani et al. 2003, Bianchi et al. 2004). An in-vitro recovery experiment was carried out and demonstrated the relative recovery of adenosine estimate to be 43.4 ± 5.1% (relative recovery was not calculated for amino acids and choline). Unlike the Swedish study patients underwent tumour resection and three microdialysis catheters were placed using an open technique: one into the tumour resection margin (T), one into peritumoural tissue 10mm away from the resection margin (PT), and one into grossly normal brain around the tumour at least 20mm away from the resection margin (BAT). Samples were analysed using various methods including HLPC. The group found that concentration of adenosine and glutamate were significantly reduced in T compared with BAT (p<0.05), the concentration of aspartate was unchanged, and the concentration of the remaining amino acids and choline were all significantly increased in T compared with BAT (p<0.01). Interestingly epilepsy, which occurs in approximately a third of patients with brain tumours (Villemure and de Tribolet 1996), was found to be an important confounding variable when

Flannery et al were the first group to take advantage of HWCO catheters to assess the cysteine protease Cathepsin S (CatS) in gliomagenesis (Flannery et al. 2007). In total 11 patients with suspected HGG were recruited. Of these 11 patients, one was subsequently found to be a low-grade glioma (LGG), 2 were cerebral metastases, and the remaining 8 cases confirmed HGGs. A further patient with suspected hydrocephalus that was undergoing intracranial pressure monitoring was also included as a control. All patients underwent tumour resection with insertion of a single microdialysis catheter at the tumour resection margin (T). Analysis of CatS was by activity and ELISA concentration assays. Unfortunately, the absence of paired catheter data makes interpretation of the study's findings difficult but there was no significant relationship between CatS concentration and

A more recent study utilising HWCO catheters in patients undergoing surgery for intrinsic brain tumours set out to first to repeat earlier measurements of glucose and its metabolites, glycerol and glutamate, and second to assess the concentration of macromolecules such as growth factors, cytokines and other proteins involved in the pathogenesis of HGGs (Marcus et al.). Eight patients with suspected HGG were recruited. Of these 8 patients, one was found to have a lymphoma, and the remaining 7 cases confirmed HGGs. A further patient

the concentrations of aspartate, glutmate and GABA were considered.

function, and the grade of brain tumours investigated.

are discussed below (see Section 3.2)

**3.2 High molecular weight cut off** 

with traumatic brain injury was included as a non-tumour control. All but one of the patients with cerebral tumours underwent surgical resection with the first microdialysis catheter placed at the tumour resection margin (T) and the second inserted at least 20mm away in macroscopically unaffected brain around tumour (BAT). The remaining one patient had an image-guided biopsy of their tumour with stereotactic insertion of a catheter into the tumour margin (T). Microdialysates were first assessed for small molecules using the CMA 600 or ISCUS analyser. Tumour microdialysates were found to have a significantly lower glucose, higher lactate/pyruvate (L/P) ratio, higher glycerol and higher glutamate compared to the brain around tumour. These findings suggest that the tumour margin of HGGs is particularly metabolically active and are consistent with previously published invitro and animal studies, but differ from the previous clinical microdialysis study by Roslin et al. There are a several reasons that may account for the discrepancy between these studies. First, the small number of cases in both studies necessitates cautious interpretation of their findings as variation in patients, their pathology and tumour heterogeneity may all have influenced the concentration of glucose and its metabolites, glycerol and glutamate. Second, Roslin et al introduced catheters following biopsy using a closed stereotactic technique while Marcus et al introduced catheters after tumour resection using an open technique. Trauma artefact may therefore have influenced findings (though in the Marcus et al study measurements were taken at least 4 hours post-operatively to try and reduce this effect). Third, while Roslin et al placed the tumour catheters in the tumour centre, Marcus et al placed tumour catheters at the tumour resection margin or tumour periphery. It is possible that the core of the tumour, which is often necrotic, may be less metabolically active than the brain-tumour interface.

In the same study Marcus et al analysed all the remaining microdialysate samples for macromolecules using a sandwich ELISA like procedure. There was great variability in the dialysate concentrations of the various growth factors (TGF-alpha, VEGF, EGF), cytokines (IL-1a, IL -1b, IL-1ra, IL-6, IL-8) and matrix metalloproteases and their tissue inhibitors (MMP2, MMP9, TIMP1, TIMP9). Nevertheless, microdialysates were found to have significantly raised MMP2/TIMP1 and IL-8 in T compared to BAT samples suggesting an environment favouring invasion and angiogenesis respectively.
