Anita Kullman

Quantitative Analysis of Tyrosine Hydroxylase mRNA for Sensitive Detection of Neuroblastoma Cells in Blood and Bone Marrow

BACKGROUND Sensitive monitoring of minimal residual disease may improve the treatment of neuroblastoma in children. To detect and monitor neuroblastoma cells in blood and bone marrow, we developed a quantitative method for the analysis of tyrosine hydroxylase mRNA. METHODS We used real-time reverse transcription-PCR. The calibrator was constructed from a segment of tyrosine hydroxylase mRNA that included the target. Blood and bone marrow samples from 24 children with neuroblastoma and 1 child with ganglioneuroma were analyzed. Controls were blood samples from the cords of 40 babies, from 58 children 6 months to 15 years of age, and from 34 healthy adults, as well as from 12 children with other diseases. RESULTS The detection limit was approximately 70 transcripts/mL. All 144 blood controls were below this limit. At diagnosis, blood tyrosine hydroxylase mRNA was higher in children with widespread disease (stage 4/4S; n = 6; range, 203-46,000 transcripts/mL) than in patients with localized disease (stages 1-3; n = 6; </=83 transcripts/mL; P = 0.002). Bone marrow from all five children with localized disease had concentrations <72 transcripts/mL, whereas five of six stage 4 patients had increased concentrations (6,000-8,000,000 transcripts/mL; P <0.05). In nine children in whom tyrosine hydroxylase mRNA was measured repeatedly, the results corresponded to the clinical course. CONCLUSION Quantitative analysis of tyrosine hydroxylase mRNA in blood and bone marrow is reliable and easy to perform and may be used for upfront staging, prognostic assessment, and treatment monitoring of neuroblastoma.

mRNAs of tyrosine hydroxylase and dopa decarboxylase but not of GD2 synthase are specific for neuroblastoma minimal disease and predicts outcome for children with high-risk disease when measured at diagnosis

Several transcripts have been claimed to be clinically valuable for detecting minimal disease in neuroblastoma, but they have not been prospectively compared in a standardized manner. Tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and GD2 synthase (GD2S) mRNAs were analyzed in 554 blood (PB) and bone marrow (BM) samples from 58 children with neuroblastoma. Samples from 44 children with other diseases served as controls. High transcript concentrations of TH, GD2S or DDC in PB or BM at diagnosis were associated with poor prognosis. TH in BM above median indicated worse outcome for a homogenous cohort with high‐risk neuroblastoma (survival probability 91% for TH below median versus 33% for TH above median, p = 0.009). The number of children with localized neuroblastoma with increased results in PB did not differ between the three transcripts. In these children, all without morphologically detectable neuroblastoma in BM, the number of patients with elevated GD2S in BM at diagnosis was significantly higher than for the other transcripts (10/16 elevated, p = 0.012). GD2S was elevated in PB from 10/28 controls without neuroblastoma compared to 1/28 for TH and DDC (p < 0.001). In BM from these children GD2S was significantly elevated. We conclude that high expression of TH and DDC both in PB and BM corresponds to metastatic neuroblastoma at diagnosis, residual disease, and poor outcome. Children with high‐risk neuroblastoma and low levels of TH in BM at diagnosis may be cured by current therapy. GD2S is less specific than TH and DDC mRNA for neuroblastoma detection in PB and BM.

Stereotactic microdialysis of the basal ganglia in Parkinson's disease

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an efficacious treatment in patients with advanced Parkinsons disease, yet the mechanisms of STN DBS are poorly understood. The aims of this study were to develop a useful method for studying neurotransmitter alterations during DBS and for the pharmacokinetics of L-dopa in brain tissue. Ten patients with Parkinsons disease participated, whereof two had no previous L-dopa medication. The electrodes and catheters were placed using MRI-guided stereotaxic targeting. Two microdialysis probes were placed, one in the right internal globus pallidus, and one in a brachial vein. The quadripolar deep brain electrodes were placed in the right STN. Microdialysates from brain tissue and blood were collected in 15-min fractions at baseline and during DBS. After stimulation new baseline fractions were taken and finally three fractions during continuous intravenous infusion of L-dopa. Clinical evaluation showed that both DBS and L-dopa infusion gave good relief of rigidity and tremor in all ten patients. During DBS the L-dopa levels in the brain increased in some of the patients but did not persist during the whole stimulation period. The concentration in brain increased substantially during intravenous L-dopa infusion. A number of catecholamines and their metabolites were analysed with high pressure liquid chromatography (HPLC). With our study we could show that this model is suitable for the monitoring of neurotransmitters and for pharmacokinetic studies in human brain, although we found that the sampling time was too short to follow the possible alterations in brain activity caused by DBS.

The Effect of Peripheral Enzyme Inhibitors on Levodopa Concentrations in Blood and CSF

Levodopa combined with a dopa‐decarboxylase inhibitor, such as carbidopa, shifts the metabolism to the COMT pathway. Adding the peripheral acting COMT inhibitor entacapone provides improvement for patients with PD suffering from motor fluctuations. We studied the effects of the enzyme inhibitors entacapone and carbidopa on the levodopa concentrations in CSF and in blood. Five PD patients with wearing‐off underwent lumbar drainage and intravenous microdialysis. Samples were taken 12 h daily for 3 days. Day 1; intravenous levodopa was given, day 2; additional oral entacapone 200 mg tid, day 3; additional oral entacapone 200 mg tid and carbidopa 25 mg bid. Levodopa in CSF and in dialysates was analysed. The AUC for levodopa increased both in blood and CSF when additional entacapone was given alone and in combination with carbidopa. The Cmax of levodopa in both CSF and blood increased significantly. Additional entacapone to levodopa therapy gives an increase of Cmax in CSF and in blood. The increase is more evident when entacapone is combined with carbidopa.

L-dopa pharmacokinetics studied with microdialysis in patients with Parkinson's disease and a history of malignant melanoma.

Objectives ‐ The pharmacokinetics of freel‐dopa in blood and tissue of five parkinsonian patients with malignant melanoma was studied with microdialysis. In one case the effect ofl‐dopa treatment on 5‐S‐cysteinyldopa and the melanoma was studied. Gastric emptying and its effects on free l‐dopa in blood were also investigated in one of the patients. Methods ‐ Five patients were given 100 mg l‐dopa with 25 mg benserazide. Blood and dialysates from the circulation and fatty tissue were collected for analysis. [13C]‐Octanoic breath test was used for analyzing gastric half‐emptying time. Results ‐ Four of the patients had similar pharmacokinetic patterns for l‐dopa and a significant (P < 0.05) increase of serum 5‐S‐cysteinyldopa occurring 30 min after l‐dopa intake. Delayed l‐dopa peaks and slow gastric half‐emptying time were found in 1 patient. A dose‐dependent increase of 5‐S‐cysteinyldopa occurred but no melanoma metastases were seen during long‐term l‐dopa therapy. Conclusion ‐ l‐dopa therapy increases 5‐S‐cysteinyldopa levels but does not seem to cause progress of melanomas. Gastric emptying impacts l‐dopa pharmacokinetics.

Comparison of N-acetylcysteine and l-2-oxothiazolidine-4-carboxylate as cysteine deliverers and glutathione precursors in human malignant melanoma transplants in mice

Purpose: Glutathione is an important cellular compound which affects detoxification of electrophiles and may have direct or indirect effects on pigment formation. It is therefore of importance to study interstitial concentrations in melanoma tissue while decreasing its formation with an enzyme inhibitor and increasing its amount with cysteine deliverers. Method: Glutathione formation was inhibited by intraperitoneal (i.p.) injection of BSO. N-Acetylcysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) were then given i.p. to subgroups of the animals. Intratumoral microdialysis was performed during BSO treatment, during BSO treatment combined with NAC or OTC and after discontinuation of BSO but ongoing NAC or OTC treatment. Results: Glutathione formation was inhibited during BSO treatment. The dialysate concentrations of both glutathione and cysteine decreased during concomitant treatment with BSO and NAC or OTC. Recovery of the amounts of the two compounds was seen in both groups after discontinuation of BSO treatment. In the NAC group we also observed an acute increase in dialysate concentrations of cysteine after NAC injection. The 5-S-cysteinyldopa concentrations were unaffected by variations in glutathione and cysteine concentrations. Conclusions: 5-S-Cysteinyldopa in melanoma is not formed from glutathione in vivo to any appreciable extent. The intracellular amount of cysteine is probably not a limiting factor for cysteinyldopa formation. It seems that both NAC and OTC can be used as cysteine deliverers to melanoma cells in vivo to produce recovery of glutathione levels after synthesis inhibition by BSO treatment.

Effects on interstitial glutathione, cysteine and 5-S-cysteinyldopa of buthionine sulphoximine in human melanoma transplants.

Using microdialysis of human melanoma transplants In athymic mice we have shown that interstitial glutathione levels decreased during treatment with buthionine sulphoximine (BSO) and recovered after cessation of treatment. The cysteine concentrations also decreased, while 5-Scysteinyldopa tended to increase during BSO treatment. Restoration of the glutathione levels was not seen after either N-acetylcysteine (NAC) or L-2-oxothiazolidine-4-carboxylate (OTC) injections, given on the third day of BSO treatment. These results were to be expected since NAC and OTC were given during the BSO treatment, and BSO is a specific and potent inhibitor of glutathione synthesis. Cysteine levels, however, increased after the NAC injection but remained unaltered after the OTC injection, while 5-Scysteinyldopa remained unaltered after both the NAC and the OTC injections.

Neurotransmitter levels in basal ganglia during levodopa and deep brain stimulation treatment in Parkinson's disease

The mechanism by which deep brain stimulation of the nucleus subthalamicus improves Parkinsons disease symptoms remains unclear. In a previous perioperative study, we showed that there might be alterations of neurotransmitter levels in the globus pallidum interna during deep brain stimulation of the nucleus subthalamicus.

Simulations and visualizations for interpretation of brain microdialysis data during deep brain stimulation

Microdialysis of the basal ganglia was used in parallel to deep brain stimulation (DBS) for patients with Parkinsons disease. The aim of this study was to patient-specifically simulate and visualize the maximum tissue volume of influence (TVImax) for each microdialysis catheter and the electric field generated around each DBS electrode. The finite element method (FEM) was used for the simulations. The method allowed mapping of the anatomical origin of the microdialysis data and the electric stimulation for each patient. It was seen that the sampling and stimulation targets differed among the patients, and the results will therefore be used in the future interpretation of the biochemical data.

Improved method for analysis of cysteinyldopa in human serum

5‐S‐l‐Cysteinyl‐l‐dopa is a well‐known pigment intermediate and analysis of its serum concentration is well suited for evaluation of treatment and follow‐up of stage III and IV malignant melanoma. A simplified analytical method is described using organic extraction followed by clean‐up on a boronate gel to capture the compound containing vicinal hydroxyls. Weak acid solution elutes the 5‐S‐cysteinyldopa suitable for high‐performance liquid chromatography (HPLC). The absolute recoveries of cysteinyldopa and its diastereomer 5‐S‐D‐cysteinyl‐l‐dopa (used as an internal standard) were 81.5±2.8% and 81.3±2.7%, respectively, and use of the internal standard for the whole procedure gave an analytical recovery of 101±0.8%. The limit of quantitation was 1.5 nmol/L and the imprecision of the method was <5.0% over the analytical range 1.5–500 nmol/L. The method is cheap and easy to perform and compares well with other described techniques. The use of the method is illustrated by results obtained during treatment of a patient with metastatic malignant melanoma.