Background The treatment for  Plasmodium falciparum  malaria in Sudan has been in process of change since 2003. Preceding the change, this study aimed to determine which artemisinin-based combination therapies is more effective to treat uncomplicated malaria in Malakal, Upper Nile, Sudan.
Background
The treatment for  Plasmodium falciparum  malaria in Sudan has been in process of change since 2003. Preceding the change, this study aimed to determine which artemisinin-based combination therapies is more effective to treat uncomplicated malaria in Malakal, Upper Nile, Sudan.Plasmodium falciparumMethods Clinical trial to assess the efficacy of 2 antimalarial therapies to treat  P. falciparum  infections in children aged 6–59 months, in a period of 42 days after treatment.
Methods
Clinical trial to assess the efficacy of 2 antimalarial therapies to treat  P. falciparum  infections in children aged 6–59 months, in a period of 42 days after treatment.P. falciparumResults A total of 269 children were followed up to 42 days. Artesunate plus Sulfadoxine/Pyrimethamine (AS+SP) and Artesunate plus Amodiaquine (AS+AQ) were both found to be efficacious in curing malaria infections by rapid elimination of parasites and clearance of fever, in preventing recrudescence and suppressing gametocytaemia. The combination of AS+SP appeared slightly more efficacious than AS+AQ, with 4.4% (4/116) versus 15% (17/113) of patients returning with malaria during the 6-week period after treatment (RR = 0.9, 95% CI 0.81–0.96). PCR analysis identified only one recrudescence which, together with one other early treatment failure, gave efficacy rates of 99.0% for AS+AQ (96/97) and 99.1% for AS+SP (112/113). However, PCR results were incomplete and assuming part of the indeterminate samples were recrudescent infections leads to an estimated efficacy ranging 97–98% for AS+SP and 88–95% for AS+AQ.
Results
A total of 269 children were followed up to 42 days. Artesunate plus Sulfadoxine/Pyrimethamine (AS+SP) and Artesunate plus Amodiaquine (AS+AQ) were both found to be efficacious in curing malaria infections by rapid elimination of parasites and clearance of fever, in preventing recrudescence and suppressing gametocytaemia. The combination of AS+SP appeared slightly more efficacious than AS+AQ, with 4.4% (4/116) versus 15% (17/113) of patients returning with malaria during the 6-week period after treatment (RR = 0.9, 95% CI 0.81–0.96). PCR analysis identified only one recrudescence which, together with one other early treatment failure, gave efficacy rates of 99.0% for AS+AQ (96/97) and 99.1% for AS+SP (112/113). However, PCR results were incomplete and assuming part of the indeterminate samples were recrudescent infections leads to an estimated efficacy ranging 97–98% for AS+SP and 88–95% for AS+AQ.Conclusion These results lead to the recommendation of ACT, and specifically AS+SP, for the treatment of uncomplicated falciparum malaria in this area of Sudan. When implemented, ACT efficacy should be monitored in sentinel sites representing different areas of the country.
Conclusion
These results lead to the recommendation of ACT, and specifically AS+SP, for the treatment of uncomplicated falciparum malaria in this area of Sudan. When implemented, ACT efficacy should be monitored in sentinel sites representing different areas of the country.

Introduction The health situation in Sudan continues to be affected by long-lasting conflict and related humanitarian emergencies such as food crises and epidemics. Malaria is one of the major causes of morbidity and mortality. In Sudan, an estimated 7.5 million patients suffer from malaria each year and 35,000 die from this disease, which accounts for up to 20% of hospital deaths [ 1 ]. The problem appears to have worsened in recent years due to increasing levels of  Plasmodium falciparum  resistance against the two most commonly used antimalarials: chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). CQ resistance in the northern and central part of the Sudan is near 50% [ 2 ]. In the South, CQ resistance below 15% was found in some isolated locations [ 3 , 4 ], but was over 80% elsewhere [ 5 , 6 ]. Resistance to SP was shown by presence of SP-resistant genotypes [ 7 ] and  in vivo  studies documenting of 0–11% resistance in Khartoum and the eastern part of the country [ 2 , 8 ] and varying levels of 0, 16% up to 70% in different locations in the southern part of the country [ 4 - 6 ]. Amodiaquine (AQ) provided an alternative for CQ in Africa in recent years; this drug is not officially registered in the Sudan, and due to its limited use, was expected to have preserved good efficacy. The nationally recommended treatment protocol for  P. falciparum  malaria in Sudan at the time of this study was still CQ as first-line and SP as second-line. A change in protocol was however in progress, and the Sudanese Health authorities had indicated that artemisinin-based combination therapy (ACT) was the preferred new first-line treatment [ 9 ]. The World Health Organization (WHO), MSF and various other NGOs supported this viewpoint [ 10 , 11 ]. In line with recommendations for several other African countries, two ACTs were proposed for Sudan: Artesunate (AS) +SP or AS+AQ. The third option, used in other African countries, Artemether-Lumefantrine (Coartem) was not seen as a viable option at the time, because of its cost. To provide information on the efficacy of ACT treatment in Sudan, the  in vivo  therapeutic efficacy of the first two combinations were studied in Malakal town, Upper Nile State.
Introduction
The health situation in Sudan continues to be affected by long-lasting conflict and related humanitarian emergencies such as food crises and epidemics. Malaria is one of the major causes of morbidity and mortality. In Sudan, an estimated 7.5 million patients suffer from malaria each year and 35,000 die from this disease, which accounts for up to 20% of hospital deaths [ 1 ]. The problem appears to have worsened in recent years due to increasing levels of  Plasmodium falciparum  resistance against the two most commonly used antimalarials: chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). CQ resistance in the northern and central part of the Sudan is near 50% [ 2 ]. In the South, CQ resistance below 15% was found in some isolated locations [ 3 , 4 ], but was over 80% elsewhere [ 5 , 6 ]. Resistance to SP was shown by presence of SP-resistant genotypes [ 7 ] and  in vivo  studies documenting of 0–11% resistance in Khartoum and the eastern part of the country [ 2 , 8 ] and varying levels of 0, 16% up to 70% in different locations in the southern part of the country [ 4 - 6 ]. Amodiaquine (AQ) provided an alternative for CQ in Africa in recent years; this drug is not officially registered in the Sudan, and due to its limited use, was expected to have preserved good efficacy.Plasmodium falciparumin vivoThe nationally recommended treatment protocol for  P. falciparum  malaria in Sudan at the time of this study was still CQ as first-line and SP as second-line. A change in protocol was however in progress, and the Sudanese Health authorities had indicated that artemisinin-based combination therapy (ACT) was the preferred new first-line treatment [ 9 ]. The World Health Organization (WHO), MSF and various other NGOs supported this viewpoint [ 10 , 11 ].P. falciparumIn line with recommendations for several other African countries, two ACTs were proposed for Sudan: Artesunate (AS) +SP or AS+AQ. The third option, used in other African countries, Artemether-Lumefantrine (Coartem) was not seen as a viable option at the time, because of its cost. To provide information on the efficacy of ACT treatment in Sudan, the  in vivo  therapeutic efficacy of the first two combinations were studied in Malakal town, Upper Nile State.in vivoPatients and Methods Malakal is located in southern Sudan (on the edge of the Khartoum-governed area), which is an area is of medium to high malaria endemicity (main transmission season: June-November). This open-label study was done in Malakal paediatric hospital supported by Médecins Sans Frontières, from September 2003 to January 2004. The methodology used was in accordance with standard WHO procedures for antimalarial drug efficacy assessment in high transmission settings [ 12 , 13 ]. Children aged between 6 months and 5 years with fever (axillary temperature of 37.5°C or more) and  P. falciparum  infection with a density of 2,000 to 200,000 parasites/μl but no signs of severe malaria (WHO criteria, [ 13 ]) or any other serious health condition were included in the study (provided written parental consent was given). One of the two therapies was allocated (by sequential alternation), and given under supervision: either (1) AS+SP, 3 days AS 4 mg/kg body weight on day 0, 1 and 2 plus SP, 25/1.25 mg/kg, single dose on day 0 or (2) AS+AQ, 3 days AS 4 mg/kg on day 0, 1 and 2 plus AQ 10 mg/kg on day 0, 1, 2. Doses were calculated for weight categories in quarter tablets. In case of vomiting within 30 minutes, the treatment was repeated. Repeated vomiting led to exclusion from study and referral to the hospital for quinine treatment. Medications were normally given in the mornings and appointments for next day roughly matched 24 hr intervals. Artesunate was obtained from Dafra (Belgium) and SP and AQ from IDA (the Netherlands). The sample size of at least 116 inclusions per arm was calculated to detect a 12% difference between the two arms, assuming a 97% efficacy at day 28 for the AS+SP arm (based on 100% efficacy of SP found in a recent study performed at a nearby location, [ 4 ]) and 85% for the AS+AQ arm (internationally recommended level for change), with 80% power and 5% risk type I error, anticipating a loss to follow-up of 10%. Patients returned daily during the first 3 days of illness and weekly thereafter for 6 weeks (42 days) after treatment. Side effects of drugs and self-medication were recorded based on patient reports and actively asked for during visits. Cases which remained negative during follow-up were considered Adequate Clinical and Parasitological Responses (ACPR). Failures were classified as (1) Early Treatment Failures (ETF) in case of significant parasitaemia at day 2 or 3 or parasites and fever at day 3, (2) Late Clinical Failures (LCF) for cases with parasites and fever during follow-up after day 3 and (3) Late Parasitological Failures (LPF) for parasite infections without fever at day 42, all in accordance with WHO guidelines [ 13 ]. Rescue treatment for failures was quinine 10 mg/kg/eight-hourly for 7 days. PCR analysis was used to distinguish new infections from recrudescences. Slides were examined by two microscopists independently and 20% of slides were cross-checked in an external laboratory (at KEMRI, Nairobi). The density of parasites was determined by simultaneous count of white blood cells and parasites, assuming a standard density of 8,000 WBCs per μl [ 13 ]. Haemoglobin levels were evaluated at day 0, 14, 28 and 42, to observe return to healthy Hb-levels after effective antimalarial treatment. PCR analysis of the  msp1  and  msp2  loci was used to distinguish re-infections from recrudescences [ 14 , 15 ]. In case of two or more identical alleles in pre- and post-treatment genotypes, the case was classified as recrudescent, or as a new infection if one or none of the alleles matched. Cases were excluded from the final analysis in case of loss to follow-up, self-medication with antimalarials and concomitant disease and when PCR results showed re-infections or were undetermined due to missing samples, undetermined genotypes or non-amplifiable DNA. The research protocol was reviewed and approved by the Sudan Research Directorate, the Malaria, Leishmaniasis and Schistosomiasis Directorate, as well as by the Ethical Review Board of MSF (comprising of MSF-external experts only). During recruitment of the children in study, informed written consent was obtained from their parents/guardians.
Patients and Methods
Malakal is located in southern Sudan (on the edge of the Khartoum-governed area), which is an area is of medium to high malaria endemicity (main transmission season: June-November). This open-label study was done in Malakal paediatric hospital supported by Médecins Sans Frontières, from September 2003 to January 2004. The methodology used was in accordance with standard WHO procedures for antimalarial drug efficacy assessment in high transmission settings [ 12 , 13 ]. Children aged between 6 months and 5 years with fever (axillary temperature of 37.5°C or more) and  P. falciparum  infection with a density of 2,000 to 200,000 parasites/μl but no signs of severe malaria (WHO criteria, [ 13 ]) or any other serious health condition were included in the study (provided written parental consent was given). One of the two therapies was allocated (by sequential alternation), and given under supervision: either (1) AS+SP, 3 days AS 4 mg/kg body weight on day 0, 1 and 2 plus SP, 25/1.25 mg/kg, single dose on day 0 or (2) AS+AQ, 3 days AS 4 mg/kg on day 0, 1 and 2 plus AQ 10 mg/kg on day 0, 1, 2. Doses were calculated for weight categories in quarter tablets. In case of vomiting within 30 minutes, the treatment was repeated. Repeated vomiting led to exclusion from study and referral to the hospital for quinine treatment. Medications were normally given in the mornings and appointments for next day roughly matched 24 hr intervals. Artesunate was obtained from Dafra (Belgium) and SP and AQ from IDA (the Netherlands).P. falciparumThe sample size of at least 116 inclusions per arm was calculated to detect a 12% difference between the two arms, assuming a 97% efficacy at day 28 for the AS+SP arm (based on 100% efficacy of SP found in a recent study performed at a nearby location, [ 4 ]) and 85% for the AS+AQ arm (internationally recommended level for change), with 80% power and 5% risk type I error, anticipating a loss to follow-up of 10%. Patients returned daily during the first 3 days of illness and weekly thereafter for 6 weeks (42 days) after treatment. Side effects of drugs and self-medication were recorded based on patient reports and actively asked for during visits. Cases which remained negative during follow-up were considered Adequate Clinical and Parasitological Responses (ACPR). Failures were classified as (1) Early Treatment Failures (ETF) in case of significant parasitaemia at day 2 or 3 or parasites and fever at day 3, (2) Late Clinical Failures (LCF) for cases with parasites and fever during follow-up after day 3 and (3) Late Parasitological Failures (LPF) for parasite infections without fever at day 42, all in accordance with WHO guidelines [ 13 ]. Rescue treatment for failures was quinine 10 mg/kg/eight-hourly for 7 days. PCR analysis was used to distinguish new infections from recrudescences.Slides were examined by two microscopists independently and 20% of slides were cross-checked in an external laboratory (at KEMRI, Nairobi). The density of parasites was determined by simultaneous count of white blood cells and parasites, assuming a standard density of 8,000 WBCs per μl [ 13 ]. Haemoglobin levels were evaluated at day 0, 14, 28 and 42, to observe return to healthy Hb-levels after effective antimalarial treatment. PCR analysis of the  msp1  and  msp2  loci was used to distinguish re-infections from recrudescences [ 14 , 15 ]. In case of two or more identical alleles in pre- and post-treatment genotypes, the case was classified as recrudescent, or as a new infection if one or none of the alleles matched. Cases were excluded from the final analysis in case of loss to follow-up, self-medication with antimalarials and concomitant disease and when PCR results showed re-infections or were undetermined due to missing samples, undetermined genotypes or non-amplifiable DNA.msp1msp2The research protocol was reviewed and approved by the Sudan Research Directorate, the Malaria, Leishmaniasis and Schistosomiasis Directorate, as well as by the Ethical Review Board of MSF (comprising of MSF-external experts only). During recruitment of the children in study, informed written consent was obtained from their parents/guardians.Results A total of 269 children were recruited during the 5-month study period. Of these, 134 were treated with AS+AQ and 135 with AS+SP. Baseline characteristics were similar in both treatment groups (Table  1 ). Of the children included, 40 were excluded from the final analysis due to associated serious other febrile illness (n = 15), self-medication with antimalarials (n = 7), loss to follow-up (n = 13) or erroneous inclusion/classification (n = 5, i.e. three cases of disagreement over microscopy quality control, two cases retreated with parasites but no fever prior to day 42 in AS+AQ group). There were three children with Hb level of 5.0 g/dl included (limit of in/exclusion), based on their condition assessed by the medical doctor. Altogether, 113 (84%) completed the study after treatment with AS+AQ and 116 (86%) with AS+SP (Figure  1 ). Table 1 Baseline characteristics of patients at study-enrolment, per treatment arm, Malakal, Upper Nile, Sudan 2003–4. AS + AQ  (n = 134) AS + SP  (n = 135) Sex ratio (F/M) 68/66 64/71 Mean age (months) (SD, ranges) 33 (13, 7–59) 33 (15, 6–59) Mean weight (kg) (SD, ranges) 11.1 (2.4, 5.7–17.0) 11.3 (2.6, 5.4–18.0) Mean axillary temperature (°C) (SD, ranges) 38.7 (0.9, 37.5–40.9) 38.9 (0.9, 37.5–40.9) Mean haemoglobin value (g/dl) (SD, ranges) 7.8 (1.7, 5.0–12.2) 7.9 (1.8, 5.0–12.7) Parasitaemia geometric mean (/μl) (ranges) 20 952 (2 100–199 500) 24835 (2 000–198 000) Figure 1 Patient flow during recruitment and follow-up Both artemisinin-combitherapies were highly effective to treat  P. falciparum  infections and prevent parasite re-emergence. Only 1 case of ETF was found, in the AS+SP group (Table  2 ). At day 28 and day 42, the proportion of patients still parasite-free was higher in the group treated with AS+SP than in the group AS +AQ (χ 2  test, p = 0.014 for results day 28; p = 0.0049 for day 42). Table 2 Treatment endpoints on day 28, 42 after treatment and PCR adjusted day 42, Malakal, Upper Nile, Sudan, 2003–2004 AS + AQ AS + SP N % 95% CI N % 95% CI Day 28 Results 117 116  ACPR 105 89.7 82.8–94.6 114 98.3 93.9 – 99.8  ETF 0 0 1 0.9 0.0–4.7  LCF 10 8.5 4.2–15.2 1 0.9 0.0–4.7 LPF 2 1.7 0.2–6.0 0 0 Day 42 Results 113 116  ACPR 96 85.0 77.0–91.0 112 96.6 91.4–99.1  ETF 0 0 1 0.9 0.0–4.7  LCF 15 13.3 7.6–20.9 2 1.7 0.2–6.1  LPF 2 1.8 0.2–6.2 1 0.9 0.0–4.7 PCR corrected day 42 # 97 113    No PCR result (12) (2)    Re-infections (4) (1)  ACPR 96 99.0 94.4–100 112 99.1 95.2–100  ETF 0 0 0.0–3.7 1 0.9 0.0–4.8  LCF 1 1.0 0.0–5.6 0 0 0.0–3.2  LPF 0 0 0.0–3.7 0 0 0.0–3.2 # Cases with missing or undetermined PCR results and re-infections were excluded from the analysis. ACPR = Adequate Clinical Parasitological Response, ETF = Early Treatment Failure, LCF = Late Clinical Failure, LPF = Late Parasitological Failure. PCR analysis was unable to generate results for a large part of cases (only 6/20 available, Figure  1 ). In the AS+AQ treatment group, of 17 failures, only 1 was confirmed to be recrudescent, whereas of the other cases, 4 were re-infections and 12 samples were not analyzable. Of the 3 'failures' in the AS + SP group, one was an early treatment failure, PCR identified 1 re-infection and 2 were non-analysable. Hence, with the PCR correction and exclusions of non-analysable and re-infection cases, AS+AQ resulted in 1.0% confirmed failures (1/97) and AS+SP in 0.9% true failures (1/113) (Table  2 ). Extrapolation of the cases with known PCR results to the ones that remained undetermined (assuming 2/3 of parasitemia due to reinfection), would give a 95.3% efficacy of AS+AQ (101/106) and 98.2% of AS+SP (112/114). Alternatively, a 'worst case' scenario, assuming that all non-analysable PCRs were actually recrudescences, would lead to 88.1% efficacy in the AS+AQ group (96/109) and 97.4% for AS+SP (111/114). Parasites were rapidly cleared in both treatment groups. At day 2 only 17% and 22% of patients still had a (low) parasitaemia in the AS+AQ and AS+SP groups, respectively. Gametocytes were found in 20% (52/262) of children on inclusion. During follow-up, 78% of these carriers at enrolment showed gametocytes again on one or more follow-up days (32/41), whereas 22% of the children without gametocytes at inclusion were later found to be gametocytemic (42/192). There was no difference between the age, temperature, parasitemia, haemoglobin on admission of the two groups, neither in rates of ACPR at study-endpoint. Gametocyte carriage was similar in both treatment groups and all gametocytes disappeared gradually from the blood during the first weeks after follow-up, and cleared by day 21 in 95% of cases (Figure  2 ). Figure 2 Parasitaemia and gametocytaemia during 6-week follow-up. Symbols: AS+SP ▲ parasites △ gametocytes, AS+AQ ● parasites ○ gametocytes. Error bars indicate 95%CI. The body temperature of all patients went down quickly; fever clearance (T<37.5°C) at day 1 and day 2 was 86% and 97% in the AS + AQ group and 83% and 93% in the AS+SP, and at day 3 all but one (on AS+SP) were free from fever. Average haemoglobin levels for the AS+AQ and AS+SP treatment groups increased from under 8.0 g/dl to near 10 g/dl 6 weeks after treatment. The proportion of children classified as moderately anaemic (haemoglobin from 5 to 8 g/dl) reduced from 51% to 11% during follow-up period, similar in both arms. No serious adverse events were observed or reported on routine clinical examination during follow-up and none of the failures developed complicated malaria.
Results
A total of 269 children were recruited during the 5-month study period. Of these, 134 were treated with AS+AQ and 135 with AS+SP. Baseline characteristics were similar in both treatment groups (Table  1 ). Of the children included, 40 were excluded from the final analysis due to associated serious other febrile illness (n = 15), self-medication with antimalarials (n = 7), loss to follow-up (n = 13) or erroneous inclusion/classification (n = 5, i.e. three cases of disagreement over microscopy quality control, two cases retreated with parasites but no fever prior to day 42 in AS+AQ group). There were three children with Hb level of 5.0 g/dl included (limit of in/exclusion), based on their condition assessed by the medical doctor. Altogether, 113 (84%) completed the study after treatment with AS+AQ and 116 (86%) with AS+SP (Figure  1 ).Baseline characteristics of patients at study-enrolment, per treatment arm, Malakal, Upper Nile, Sudan 2003–4.AS + AQAS + SPBoth artemisinin-combitherapies were highly effective to treat  P. falciparum  infections and prevent parasite re-emergence. Only 1 case of ETF was found, in the AS+SP group (Table  2 ). At day 28 and day 42, the proportion of patients still parasite-free was higher in the group treated with AS+SP than in the group AS +AQ (χ 2  test, p = 0.014 for results day 28; p = 0.0049 for day 42).P. falciparumTreatment endpoints on day 28, 42 after treatment and PCR adjusted day 42, Malakal, Upper Nile, Sudan, 2003–2004AS + AQAS + SPDay 28 Results11711689.798.300.98.50.91.70Day 42 Results11311685.096.600.913.31.71.80.9PCR corrected day 4297113No PCR result(12)(2)Re-infections(4)(1)99.099.100.91.0000# Cases with missing or undetermined PCR results and re-infections were excluded from the analysis. ACPR = Adequate Clinical Parasitological Response, ETF = Early Treatment Failure, LCF = Late Clinical Failure, LPF = Late Parasitological Failure.PCR analysis was unable to generate results for a large part of cases (only 6/20 available, Figure  1 ). In the AS+AQ treatment group, of 17 failures, only 1 was confirmed to be recrudescent, whereas of the other cases, 4 were re-infections and 12 samples were not analyzable. Of the 3 'failures' in the AS + SP group, one was an early treatment failure, PCR identified 1 re-infection and 2 were non-analysable. Hence, with the PCR correction and exclusions of non-analysable and re-infection cases, AS+AQ resulted in 1.0% confirmed failures (1/97) and AS+SP in 0.9% true failures (1/113) (Table  2 ). Extrapolation of the cases with known PCR results to the ones that remained undetermined (assuming 2/3 of parasitemia due to reinfection), would give a 95.3% efficacy of AS+AQ (101/106) and 98.2% of AS+SP (112/114). Alternatively, a 'worst case' scenario, assuming that all non-analysable PCRs were actually recrudescences, would lead to 88.1% efficacy in the AS+AQ group (96/109) and 97.4% for AS+SP (111/114).Parasites were rapidly cleared in both treatment groups. At day 2 only 17% and 22% of patients still had a (low) parasitaemia in the AS+AQ and AS+SP groups, respectively. Gametocytes were found in 20% (52/262) of children on inclusion. During follow-up, 78% of these carriers at enrolment showed gametocytes again on one or more follow-up days (32/41), whereas 22% of the children without gametocytes at inclusion were later found to be gametocytemic (42/192). There was no difference between the age, temperature, parasitemia, haemoglobin on admission of the two groups, neither in rates of ACPR at study-endpoint. Gametocyte carriage was similar in both treatment groups and all gametocytes disappeared gradually from the blood during the first weeks after follow-up, and cleared by day 21 in 95% of cases (Figure  2 ).The body temperature of all patients went down quickly; fever clearance (T<37.5°C) at day 1 and day 2 was 86% and 97% in the AS + AQ group and 83% and 93% in the AS+SP, and at day 3 all but one (on AS+SP) were free from fever. Average haemoglobin levels for the AS+AQ and AS+SP treatment groups increased from under 8.0 g/dl to near 10 g/dl 6 weeks after treatment. The proportion of children classified as moderately anaemic (haemoglobin from 5 to 8 g/dl) reduced from 51% to 11% during follow-up period, similar in both arms. No serious adverse events were observed or reported on routine clinical examination during follow-up and none of the failures developed complicated malaria.Discussion This is one of the first studies into the clinical outcomes of ACT combination therapy in Sudan and provides useful information for decision-makers working to ensure effective antimalarial protocols in this part of the country. Both of the artemisinin-based combination therapies tested here were found to be highly efficacious in the treatment of uncomplicated  P. falciparum  malaria in this area of the Sudan. AS+SP appears to be the better treatment option on the basis of non-PCR corrected responses, showing a lower percentage of patients returning with parasitaemia. The PCR analysis indicates the true efficacy is comparable between both treatments (near 99%), but is, however, limited by a high proportion of indeterminate cases. More realistically, an efficacy between 97–98% can be expected for AS+SP and 88–95% for AS+AQ, acceptable levels after the long follow-up of 42 days after treatment. A rapid parasite clearance and fever reduction was found following treatment with both ACTs. The rise in haemoglobin values and the reduction of the proportion of (moderately) anaemic children after treatment confirms that the malaria parasites were effectively removed from the blood and red blood cell levels rose after treatment. The ACTs tested also had an effect on gametocytes. In general, the gametocidal action of AS appears to work through preventing the development of new gametocytes rather than clearance of existing ones [ 16 ]. In our study, the 20% of gametocyte-carrying infections at enrolment cleared gradually by day 21. Newly detected gametocytes developed in 22% of cases after treatment, which is lower than after monotherapy, at least for SP. A previous study in Upper Nile [ 4 ] showed that 68% of patients (of all ages) treated with SP and 28% of those treated with AQ developed gametocytaemia during the 14 days after treatment, while gametocyte prevalence at admission was only 2%. Gametocidal effect is very important since the sexual stages of parasites are essential for person-to-person transmission of malaria via mosquito vectors. The main limitations of the study were that the number of patient exclusions were higher than anticipated (15%) due to concomitant febrile illnesses and loss to follow-up, as well as the lack of results for PCR-analysis, caused by samples missing or an inability to provide sufficient DNA on amplification due to low densities in post-treatment samples. Increasing the number of inclusions compensated losses to follow-up. Repeated attempts at PCR-analysis for problematic samples were only partly successful and, therefore, lead to extrapolation about the findings for the missing samples. At time of writing, a change of national protocols towards ACT in northern Sudan is in preparation – coordinated among health authorities, NGOs and other relevant actors. The first line treatment recommended country-wide is AS+SP, based on reported high efficacy of SP. Northern Sudan is the only country in Africa, which has chosen the option AS+SP [ 17 ]. On the basis of the results of this study, this is justifiable. One other study in Sudan on AS+SP and AS+AQ efficacy was recently completed in the Nuba Mountains and also shows high efficacy of 91.2% and 92.7%, respectively, for these ACTs at 28 days after treatment [ 18 ]. ACTs with SP or AQ as companion drugs have shown to be very effective in other areas of Africa, provided that the companion drug still maintained a good level of efficacy [ 19 - 22 ]. In the Upper Nile area both SP and AQ were still an effective treatment for  P. falciparum  and SP has shown high efficacy in various areas of northern Sudan [ 2 , 4 , 23 ]. It remains to be seen whether AS+SP will be equally efficacious in other, e.g. more northern areas of the Sudan. In Southern Sudan AS+AQ has recently been put forward as the therapy of choice [ 17 ]. The use of two different therapies would seem a sensible option in a vast country with areas of different patterns of resistance. An advantage of AS+SP over AS+AQ is that it is more convenient to administer, as SP is given as a single dose and can be administered under observation in a health facility, whereas AQ requires 3 days to complete a course. SP tablets are also easier to take (AQ has a bitter taste). Blister packs of AS+SP, which combine the two drugs and clearly indicate daily tablets to be taken, are currently available for different age categories in Sudan. AS+SP is cheaper than AS+AQ. Implementation of the new national protocol with AS+SP will hopefully take place as quickly as possible to prevent rising morbidity and mortality. Vulnerable displaced populations in epidemic-prone areas and areas of high perennial malaria transmission should be prioritized. Introduction of ACT will have to go hand in hand with laboratory confirmed diagnosis (microscopy or rapid diagnostic tests) to prevent unnecessary use of valuable drugs (thus minimizing drug pressure) and ensure that non-malarial cases are appropriately treated. The change in guidelines should also filter through all health service providers, including the private sector and drug vendors to decrease the potential risk of SP monotherapy and incomplete dosages. The change will initially require more funds to be made available for malaria treatment. The international community – including many NGOs, the WHO, donors, and the Global Fund – has shown willingness to support countries to change antimalarial protocols [ 24 , 25 ]. At present, MSF has already started to pilot AS+SP treatment in its project areas in Northern Sudan, i.e. Darfur, Upper Nile and Gedaref, on behalf of the Ministry of Health. The implementation of treatment as well as the future efficacy of AS+SP should be monitored carefully in a number of dispersed sentinel sites, as there is a possibility that SP resistance may further rise before the combination has been made available countrywide.
Discussion
This is one of the first studies into the clinical outcomes of ACT combination therapy in Sudan and provides useful information for decision-makers working to ensure effective antimalarial protocols in this part of the country.Both of the artemisinin-based combination therapies tested here were found to be highly efficacious in the treatment of uncomplicated  P. falciparum  malaria in this area of the Sudan. AS+SP appears to be the better treatment option on the basis of non-PCR corrected responses, showing a lower percentage of patients returning with parasitaemia. The PCR analysis indicates the true efficacy is comparable between both treatments (near 99%), but is, however, limited by a high proportion of indeterminate cases. More realistically, an efficacy between 97–98% can be expected for AS+SP and 88–95% for AS+AQ, acceptable levels after the long follow-up of 42 days after treatment. A rapid parasite clearance and fever reduction was found following treatment with both ACTs. The rise in haemoglobin values and the reduction of the proportion of (moderately) anaemic children after treatment confirms that the malaria parasites were effectively removed from the blood and red blood cell levels rose after treatment.P. falciparumThe ACTs tested also had an effect on gametocytes. In general, the gametocidal action of AS appears to work through preventing the development of new gametocytes rather than clearance of existing ones [ 16 ]. In our study, the 20% of gametocyte-carrying infections at enrolment cleared gradually by day 21. Newly detected gametocytes developed in 22% of cases after treatment, which is lower than after monotherapy, at least for SP. A previous study in Upper Nile [ 4 ] showed that 68% of patients (of all ages) treated with SP and 28% of those treated with AQ developed gametocytaemia during the 14 days after treatment, while gametocyte prevalence at admission was only 2%. Gametocidal effect is very important since the sexual stages of parasites are essential for person-to-person transmission of malaria via mosquito vectors.The main limitations of the study were that the number of patient exclusions were higher than anticipated (15%) due to concomitant febrile illnesses and loss to follow-up, as well as the lack of results for PCR-analysis, caused by samples missing or an inability to provide sufficient DNA on amplification due to low densities in post-treatment samples. Increasing the number of inclusions compensated losses to follow-up. Repeated attempts at PCR-analysis for problematic samples were only partly successful and, therefore, lead to extrapolation about the findings for the missing samples.At time of writing, a change of national protocols towards ACT in northern Sudan is in preparation – coordinated among health authorities, NGOs and other relevant actors. The first line treatment recommended country-wide is AS+SP, based on reported high efficacy of SP. Northern Sudan is the only country in Africa, which has chosen the option AS+SP [ 17 ]. On the basis of the results of this study, this is justifiable. One other study in Sudan on AS+SP and AS+AQ efficacy was recently completed in the Nuba Mountains and also shows high efficacy of 91.2% and 92.7%, respectively, for these ACTs at 28 days after treatment [ 18 ]. ACTs with SP or AQ as companion drugs have shown to be very effective in other areas of Africa, provided that the companion drug still maintained a good level of efficacy [ 19 - 22 ]. In the Upper Nile area both SP and AQ were still an effective treatment for  P. falciparum  and SP has shown high efficacy in various areas of northern Sudan [ 2 , 4 , 23 ]. It remains to be seen whether AS+SP will be equally efficacious in other, e.g. more northern areas of the Sudan. In Southern Sudan AS+AQ has recently been put forward as the therapy of choice [ 17 ]. The use of two different therapies would seem a sensible option in a vast country with areas of different patterns of resistance.P. falciparumAn advantage of AS+SP over AS+AQ is that it is more convenient to administer, as SP is given as a single dose and can be administered under observation in a health facility, whereas AQ requires 3 days to complete a course. SP tablets are also easier to take (AQ has a bitter taste). Blister packs of AS+SP, which combine the two drugs and clearly indicate daily tablets to be taken, are currently available for different age categories in Sudan. AS+SP is cheaper than AS+AQ.Implementation of the new national protocol with AS+SP will hopefully take place as quickly as possible to prevent rising morbidity and mortality. Vulnerable displaced populations in epidemic-prone areas and areas of high perennial malaria transmission should be prioritized. Introduction of ACT will have to go hand in hand with laboratory confirmed diagnosis (microscopy or rapid diagnostic tests) to prevent unnecessary use of valuable drugs (thus minimizing drug pressure) and ensure that non-malarial cases are appropriately treated. The change in guidelines should also filter through all health service providers, including the private sector and drug vendors to decrease the potential risk of SP monotherapy and incomplete dosages. The change will initially require more funds to be made available for malaria treatment. The international community – including many NGOs, the WHO, donors, and the Global Fund – has shown willingness to support countries to change antimalarial protocols [ 24 , 25 ].At present, MSF has already started to pilot AS+SP treatment in its project areas in Northern Sudan, i.e. Darfur, Upper Nile and Gedaref, on behalf of the Ministry of Health. The implementation of treatment as well as the future efficacy of AS+SP should be monitored carefully in a number of dispersed sentinel sites, as there is a possibility that SP resistance may further rise before the combination has been made available countrywide.Authors' contributions IB was responsible for overall supervision, data analysis and writing of the paper, RA for field-implementation, communication as well as data analysis and writing, MB for preparation and set-up of study, support and advocacy. PH was responsible for microscopy laboratory work and quality. EA participated as field supervisor of clinical work and data gathering. EBH and MAF were responsible for day-to-day study procedures and supervision of clinical teams. JM did the PCR analysis and FC was involved in design of study, data analysis and writing of manuscript. All authors read and approved the final manuscript.
Authors' contributions
IB was responsible for overall supervision, data analysis and writing of the paper, RA for field-implementation, communication as well as data analysis and writing, MB for preparation and set-up of study, support and advocacy. PH was responsible for microscopy laboratory work and quality. EA participated as field supervisor of clinical work and data gathering. EBH and MAF were responsible for day-to-day study procedures and supervision of clinical teams. JM did the PCR analysis and FC was involved in design of study, data analysis and writing of manuscript. All authors read and approved the final manuscript.


Background Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide.
Background
Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide.Methods We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas [ 1 ]. These profiles were produced as part of the Cancer Genome Anatomy Project's  SAGE Genie  [ 2 ], and were used in an  in silico  search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors.
Methods
We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas [ 1 ]. These profiles were produced as part of the Cancer Genome Anatomy Project's  SAGE Genie  [ 2 ], and were used in an  in silico  search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors.SAGE Geniein silicoResults A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions.
Results
A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions.Conclusions This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes.
Conclusions
This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes.

Background Astrocytomas are the most frequent malignant primary brain tumors in adults. Clinically, this group of tumors can be divided into four World Health Organization (WHO) grades. Pilocytic astrocytomas (WHO grade I) are generally slow growing and non-infiltrative pediatric tumors, which are rarely fatal. For the infiltrating astrocytomas, survival decreases with increasing grade. Grade II astrocytomas patients survive an average of over 5 years, but survival drops to 3 years for anaplastic astrocytomas (grade III). Grade IV astrocytomas (glioblastoma multiforme or GBM) account for about half of all astrocytic tumors, with a median survival of less than a year. Effective treatment options for the invasive grade II to IV tumors are still limited to surgery and radiation therapy, with most chemotherapy regimens showing little or no increase in survival. Several recent gene expression profiling studies of human astrocytomas have been able to distinguish between various grades of astrocytomas and between astrocytomas and other human glial tumors, and to identify new molecular classes within histological grade [ 3 - 7 ]. This enhanced molecular classification based on expression patterns of genes and pathways holds promise for better diagnostic and prognostic tools. Candidate glioblastoma associated genes have also been identified using expression profiling [ 8 - 11 ]. While these studies in brain cancers have produced leads for potential therapeutic targets, a systematic and comprehensive evaluation of gene expression in malignant astrocytomas is not readily and freely available for the scientific community. In this study, we sought to create a public and comprehensive gene expression resource for astrocytomas, with the primary intention of aiding searches for new therapeutic targets in malignant astrocytomas. For this purpose we produced and analyzed in detail 25 gene expression profiles of primary astrocytic tumors (grade II, III and IV) using Serial Analysis of Gene Expression (SAGE) [ 12 ]. Complete expression profiles are posted for the scientific community at the CGAP SAGE Genie website  [ 2 ]. The utility of the resource was validated by extensive comparisons of tumor with normal tissue. SAGE profiles on normal brain and other tissues created by the Cancer Genome Anatomy Project [ 8 ] were used to subtract out the genes normally expressed in adult brain, leaving a small and specific set of astrocytoma associated genes for each class, and revealing cell surface or extra-cellular matrix related genes highly expressed in the tumors when compared to their expression in normal tissue. A subset of the tumor-associated genes was validated in an independent set of tumors at both the transcript and protein level. In summary we have identified several novel tumor-associated markers in astrocytic tumors as well as various cell surface markers highly expressed in the most aggressive tumor types.
Background
Astrocytomas are the most frequent malignant primary brain tumors in adults. Clinically, this group of tumors can be divided into four World Health Organization (WHO) grades. Pilocytic astrocytomas (WHO grade I) are generally slow growing and non-infiltrative pediatric tumors, which are rarely fatal. For the infiltrating astrocytomas, survival decreases with increasing grade. Grade II astrocytomas patients survive an average of over 5 years, but survival drops to 3 years for anaplastic astrocytomas (grade III). Grade IV astrocytomas (glioblastoma multiforme or GBM) account for about half of all astrocytic tumors, with a median survival of less than a year. Effective treatment options for the invasive grade II to IV tumors are still limited to surgery and radiation therapy, with most chemotherapy regimens showing little or no increase in survival.Several recent gene expression profiling studies of human astrocytomas have been able to distinguish between various grades of astrocytomas and between astrocytomas and other human glial tumors, and to identify new molecular classes within histological grade [ 3 - 7 ]. This enhanced molecular classification based on expression patterns of genes and pathways holds promise for better diagnostic and prognostic tools. Candidate glioblastoma associated genes have also been identified using expression profiling [ 8 - 11 ]. While these studies in brain cancers have produced leads for potential therapeutic targets, a systematic and comprehensive evaluation of gene expression in malignant astrocytomas is not readily and freely available for the scientific community.In this study, we sought to create a public and comprehensive gene expression resource for astrocytomas, with the primary intention of aiding searches for new therapeutic targets in malignant astrocytomas. For this purpose we produced and analyzed in detail 25 gene expression profiles of primary astrocytic tumors (grade II, III and IV) using Serial Analysis of Gene Expression (SAGE) [ 12 ]. Complete expression profiles are posted for the scientific community at the CGAP SAGE Genie website  [ 2 ]. The utility of the resource was validated by extensive comparisons of tumor with normal tissue. SAGE profiles on normal brain and other tissues created by the Cancer Genome Anatomy Project [ 8 ] were used to subtract out the genes normally expressed in adult brain, leaving a small and specific set of astrocytoma associated genes for each class, and revealing cell surface or extra-cellular matrix related genes highly expressed in the tumors when compared to their expression in normal tissue. A subset of the tumor-associated genes was validated in an independent set of tumors at both the transcript and protein level. In summary we have identified several novel tumor-associated markers in astrocytic tumors as well as various cell surface markers highly expressed in the most aggressive tumor types.Methods Tissue and RNA Astrocytic tumor samples from 21 adults and 4 children were obtained from the Duke Brain Tumor Bank. All samples were classified based on histology according to the World Heath Organization grading criteria. Pediatric normal cortex (15 months) was a gift of Dr. Rachel Myerowitz and normal pediatric cerebellum was from the Maryland Brain Bank. Normal adult cortex and cerebellum were rapid autopsy samples obtained from the Duke Alzheimer's Brain Bank. Total RNA from normal substantia nigra was obtained from Clontech (Palo Alto, CA). PolyA+ RNA from normal adult leukocytes was obtained from Stratagene (La Jolla, CA), as noted for each library's information at the SAGE Genie Website [ 2 ]. RNA integrity was confirmed by gel electrophoresis prior to SAGE library construction. SAGE libraries and informatics SAGE libraries from 25 selected astrocytomas: 8 grade II astrocytomas, 10 grade III anaplastic astrocytomas and 7 grade IV glioblastomas (primary GBM) were constructed using  Nla  III as the anchoring enzyme and  BsmF  I as the tagging enzyme using a micro-SAGE protocol. The SAGE library clones were partially arrayed at Lawrence Livermore National Laboratories and inserts were purified and sequenced at the BC Cancer Agency Genome Sciences Centre or arrayed and sequenced at Agencourt Bioscience Corporation. The SAGE 2000 software version 4.12 (available at  ) was used to extract SAGE tags from the original sequence files, remove duplicate ditags, remove linker sequences, remove one base pair variations of linker sequences and tabulate the occurrence of each tag. Tag sequences, tag counts and gene associations were stored in a Microsoft Access relational database for subsequent selection of tags with a particular profile. A total of 2,605,122 tags were obtained with an average of 102,988 tags per library. Normal neural tissue tags included a total of 443,560 tags from normal brain [ 8 , 13 ]. These SAGE normal brain libraries have the following unique identifiers: cortex_B_BB542; cortex_B_pool6; thalamus_B_1; cerebellum_B_1; cerebellum_B_BB542; peds_cortex_B_H1571 and substantia_nigra_B_1. Tags totaling 48,039 were also included from normal leukocytes (SAGE_Leukocytes_normal_B_1). Detailed library information and tag counts for each tissue are located at CGAP's SAGE Genie  [ 2 ]. Tag counts were normalized to 100,000 tags per library. Real-time PCR Total RNA extraction, cDNA synthesis and quantitative PCR were performed as previously described [ 1 ]. Gene expression levels were normalized to 3 genes; GAPDH, ribosomal protein RPS27 and low molecular mass ubiquinone-binding protein (QP_C). Both RPS27 and QP-C showed a relatively even expression level across the libraries as assed by SAGE analysis. Relative expression levels were calculated in comparison to the levels in nine normal neural tissues, including normal brain (3x), cerebellum (2x), thalamus, gray matter, caudate nucleus and pediatric cortex according to Saha  et al ., 2001 [ 14 ]. A list of the PCR primers used for each gene is available upon request. Immunohistochemistry Formalin-fixed 5-μm paraffin-embedded sections were stained with various antibodies using the biotin/streptavidin RTU Vectastain Universal Quick kit (Vector laboratories, Burlingame, CA) as previously described [ 1 ]. Shortly, sections were deparaffinized in HemoD and re-hydrated through descending alcohols. Endogenous peroxidase was quenched by incubating the slides in methanol/5% H 2 O 2  at room temperature for 10 min. Non-enzymatic antigen retrieval was performed using the Antigen retrieval solution AR or Citra (Biogenex, San Ramon, CA) in combination with microwave treatment. Sections were then blocked in PBS/0.5% Triton X-100 containing 2.5% normal horse serum for at least 30 min at room temperature and incubated with primary antibody overnight at 4°C. The primary antibodies used were mouse monoclonal anti-aquaporin1, at a 1:60 dilution (Abcam Limited, Cambridgeshire, United Kingdom) and mouse monoclonal anti-Topoisomerase II Alpha used at a 1:30 dilution (Novocatra Laboratories, New Castle, United Kingdom). Sections were developed with a DAB (Sigma, St. Louis, MO) substrate, counterstained with hematoxylin and mounted with Cytoseal-60. Tissue micro arrays The tissue micro array used in these studies contained cores from 20 glioblastomas, 20 anaplastic astrocytomas, 20 infiltrating astrocytomas and 20 oligodendroglial lesions and was prepared according to methods described by Kononen and Kallioniemi [ 15 ]. Microscopic examination of the array confirmed that the appearance of the tumor tissue cores corresponded to that in the donor blocks. A neuropathologist (CGE) selected the tumor areas sampled in each case and examined the resulting arrays to ensure they accurately represented the donor cases.
Methods
Tissue and RNA Astrocytic tumor samples from 21 adults and 4 children were obtained from the Duke Brain Tumor Bank. All samples were classified based on histology according to the World Heath Organization grading criteria. Pediatric normal cortex (15 months) was a gift of Dr. Rachel Myerowitz and normal pediatric cerebellum was from the Maryland Brain Bank. Normal adult cortex and cerebellum were rapid autopsy samples obtained from the Duke Alzheimer's Brain Bank. Total RNA from normal substantia nigra was obtained from Clontech (Palo Alto, CA). PolyA+ RNA from normal adult leukocytes was obtained from Stratagene (La Jolla, CA), as noted for each library's information at the SAGE Genie Website [ 2 ]. RNA integrity was confirmed by gel electrophoresis prior to SAGE library construction.
Tissue and RNA
Astrocytic tumor samples from 21 adults and 4 children were obtained from the Duke Brain Tumor Bank. All samples were classified based on histology according to the World Heath Organization grading criteria. Pediatric normal cortex (15 months) was a gift of Dr. Rachel Myerowitz and normal pediatric cerebellum was from the Maryland Brain Bank. Normal adult cortex and cerebellum were rapid autopsy samples obtained from the Duke Alzheimer's Brain Bank. Total RNA from normal substantia nigra was obtained from Clontech (Palo Alto, CA). PolyA+ RNA from normal adult leukocytes was obtained from Stratagene (La Jolla, CA), as noted for each library's information at the SAGE Genie Website [ 2 ]. RNA integrity was confirmed by gel electrophoresis prior to SAGE library construction.SAGE libraries and informatics SAGE libraries from 25 selected astrocytomas: 8 grade II astrocytomas, 10 grade III anaplastic astrocytomas and 7 grade IV glioblastomas (primary GBM) were constructed using  Nla  III as the anchoring enzyme and  BsmF  I as the tagging enzyme using a micro-SAGE protocol. The SAGE library clones were partially arrayed at Lawrence Livermore National Laboratories and inserts were purified and sequenced at the BC Cancer Agency Genome Sciences Centre or arrayed and sequenced at Agencourt Bioscience Corporation. The SAGE 2000 software version 4.12 (available at  ) was used to extract SAGE tags from the original sequence files, remove duplicate ditags, remove linker sequences, remove one base pair variations of linker sequences and tabulate the occurrence of each tag. Tag sequences, tag counts and gene associations were stored in a Microsoft Access relational database for subsequent selection of tags with a particular profile. A total of 2,605,122 tags were obtained with an average of 102,988 tags per library. Normal neural tissue tags included a total of 443,560 tags from normal brain [ 8 , 13 ]. These SAGE normal brain libraries have the following unique identifiers: cortex_B_BB542; cortex_B_pool6; thalamus_B_1; cerebellum_B_1; cerebellum_B_BB542; peds_cortex_B_H1571 and substantia_nigra_B_1. Tags totaling 48,039 were also included from normal leukocytes (SAGE_Leukocytes_normal_B_1). Detailed library information and tag counts for each tissue are located at CGAP's SAGE Genie  [ 2 ]. Tag counts were normalized to 100,000 tags per library.
SAGE libraries and informatics
SAGE libraries from 25 selected astrocytomas: 8 grade II astrocytomas, 10 grade III anaplastic astrocytomas and 7 grade IV glioblastomas (primary GBM) were constructed using  Nla  III as the anchoring enzyme and  BsmF  I as the tagging enzyme using a micro-SAGE protocol. The SAGE library clones were partially arrayed at Lawrence Livermore National Laboratories and inserts were purified and sequenced at the BC Cancer Agency Genome Sciences Centre or arrayed and sequenced at Agencourt Bioscience Corporation.NlaBsmFThe SAGE 2000 software version 4.12 (available at  ) was used to extract SAGE tags from the original sequence files, remove duplicate ditags, remove linker sequences, remove one base pair variations of linker sequences and tabulate the occurrence of each tag. Tag sequences, tag counts and gene associations were stored in a Microsoft Access relational database for subsequent selection of tags with a particular profile. A total of 2,605,122 tags were obtained with an average of 102,988 tags per library. Normal neural tissue tags included a total of 443,560 tags from normal brain [ 8 , 13 ]. These SAGE normal brain libraries have the following unique identifiers: cortex_B_BB542; cortex_B_pool6; thalamus_B_1; cerebellum_B_1; cerebellum_B_BB542; peds_cortex_B_H1571 and substantia_nigra_B_1. Tags totaling 48,039 were also included from normal leukocytes (SAGE_Leukocytes_normal_B_1). Detailed library information and tag counts for each tissue are located at CGAP's SAGE Genie  [ 2 ]. Tag counts were normalized to 100,000 tags per library.Real-time PCR Total RNA extraction, cDNA synthesis and quantitative PCR were performed as previously described [ 1 ]. Gene expression levels were normalized to 3 genes; GAPDH, ribosomal protein RPS27 and low molecular mass ubiquinone-binding protein (QP_C). Both RPS27 and QP-C showed a relatively even expression level across the libraries as assed by SAGE analysis. Relative expression levels were calculated in comparison to the levels in nine normal neural tissues, including normal brain (3x), cerebellum (2x), thalamus, gray matter, caudate nucleus and pediatric cortex according to Saha  et al ., 2001 [ 14 ]. A list of the PCR primers used for each gene is available upon request.
Real-time PCR
Total RNA extraction, cDNA synthesis and quantitative PCR were performed as previously described [ 1 ]. Gene expression levels were normalized to 3 genes; GAPDH, ribosomal protein RPS27 and low molecular mass ubiquinone-binding protein (QP_C). Both RPS27 and QP-C showed a relatively even expression level across the libraries as assed by SAGE analysis. Relative expression levels were calculated in comparison to the levels in nine normal neural tissues, including normal brain (3x), cerebellum (2x), thalamus, gray matter, caudate nucleus and pediatric cortex according to Saha  et al ., 2001 [ 14 ]. A list of the PCR primers used for each gene is available upon request.et alImmunohistochemistry Formalin-fixed 5-μm paraffin-embedded sections were stained with various antibodies using the biotin/streptavidin RTU Vectastain Universal Quick kit (Vector laboratories, Burlingame, CA) as previously described [ 1 ]. Shortly, sections were deparaffinized in HemoD and re-hydrated through descending alcohols. Endogenous peroxidase was quenched by incubating the slides in methanol/5% H 2 O 2  at room temperature for 10 min. Non-enzymatic antigen retrieval was performed using the Antigen retrieval solution AR or Citra (Biogenex, San Ramon, CA) in combination with microwave treatment. Sections were then blocked in PBS/0.5% Triton X-100 containing 2.5% normal horse serum for at least 30 min at room temperature and incubated with primary antibody overnight at 4°C. The primary antibodies used were mouse monoclonal anti-aquaporin1, at a 1:60 dilution (Abcam Limited, Cambridgeshire, United Kingdom) and mouse monoclonal anti-Topoisomerase II Alpha used at a 1:30 dilution (Novocatra Laboratories, New Castle, United Kingdom). Sections were developed with a DAB (Sigma, St. Louis, MO) substrate, counterstained with hematoxylin and mounted with Cytoseal-60.
Immunohistochemistry
Formalin-fixed 5-μm paraffin-embedded sections were stained with various antibodies using the biotin/streptavidin RTU Vectastain Universal Quick kit (Vector laboratories, Burlingame, CA) as previously described [ 1 ]. Shortly, sections were deparaffinized in HemoD and re-hydrated through descending alcohols. Endogenous peroxidase was quenched by incubating the slides in methanol/5% H 2 O 2  at room temperature for 10 min. Non-enzymatic antigen retrieval was performed using the Antigen retrieval solution AR or Citra (Biogenex, San Ramon, CA) in combination with microwave treatment. Sections were then blocked in PBS/0.5% Triton X-100 containing 2.5% normal horse serum for at least 30 min at room temperature and incubated with primary antibody overnight at 4°C. The primary antibodies used were mouse monoclonal anti-aquaporin1, at a 1:60 dilution (Abcam Limited, Cambridgeshire, United Kingdom) and mouse monoclonal anti-Topoisomerase II Alpha used at a 1:30 dilution (Novocatra Laboratories, New Castle, United Kingdom). Sections were developed with a DAB (Sigma, St. Louis, MO) substrate, counterstained with hematoxylin and mounted with Cytoseal-60.Tissue micro arrays The tissue micro array used in these studies contained cores from 20 glioblastomas, 20 anaplastic astrocytomas, 20 infiltrating astrocytomas and 20 oligodendroglial lesions and was prepared according to methods described by Kononen and Kallioniemi [ 15 ]. Microscopic examination of the array confirmed that the appearance of the tumor tissue cores corresponded to that in the donor blocks. A neuropathologist (CGE) selected the tumor areas sampled in each case and examined the resulting arrays to ensure they accurately represented the donor cases.
Tissue micro arrays
The tissue micro array used in these studies contained cores from 20 glioblastomas, 20 anaplastic astrocytomas, 20 infiltrating astrocytomas and 20 oligodendroglial lesions and was prepared according to methods described by Kononen and Kallioniemi [ 15 ]. Microscopic examination of the array confirmed that the appearance of the tumor tissue cores corresponded to that in the donor blocks. A neuropathologist (CGE) selected the tumor areas sampled in each case and examined the resulting arrays to ensure they accurately represented the donor cases.Results and Discussion SAGE gene expression profiles, selection and confirmation of tumor-associated genes This report describes the comprehensive generation of expression profiles of three astrocytic tumor grades based on Serial Analysis of Gene Expression (SAGE). The main goals were to identify genes not expressed in normal brain tissue and genes highly expressed in the more aggressive astrocytomas encoding cell surface or extra-cellular matrix related proteins that could be of potential therapeutic interest. We generated SAGE profiles on 8 infiltrating astrocytomas, 10 anaplastic astrocytomas and 7 glioblastoma samples. Combined with two glioblastoma profiles [ 8 ], previously deposited on SAGE Genie, this study analyzed 2,734,106 astrocytoma SAGE tags. On average we could distinguish over 27,000 unique tags in each tumor grade after excluding those with single counts (Table  1 ). Complete expression profiles and library information are posted for the scientific community at the Cancer Genome Anatomy Project (CGAP) SAGE Genie website  , where the libraries can be downloaded or viewed online using SAGE Genie tools [ 2 ]. In order to identify tumor-associated genes, we sought highly expressed transcripts in each grade of astrocytoma that were not expressed in 7 normal brain tissues. This also helped control for contaminating normal cells within the tumor sample. Tags expressed in a normal leukocyte SAGE library were also included in the analysis so they could be subtracted to reduce the chances of identifying transcripts from white blood cells that frequently infiltrate these tumors. Initially we selected for tags with an average expression of at least 3 per 100,000 tags. Subsequently, we selected for tags expressed at less than 2 counts per 100,000 in each of the 8 normal libraries (7 normal neural tissues & one leukocyte), reducing the number of tags to less than 100 per tumor type (Table  1 ). We further narrowed down the list of candidates by including only those tag sequences that could be matched with a full-length cDNA sequence. From these lists of genes we selected respectively 8, 16 and 10 genes for real-time PCR analysis in an independent set of 14 to 17 grade II, grade III and grade IV primary tumors. Only 6 genes could not be confirmed by real-time PCR analysis, 3 of 8 (grade II selection) and 3 of 16 (grade III selection). Another 8 genes were confirmed in only 20 to 25 % of the tumors. Table  2  lists those genes with a 5-fold or more over-expression by real time PCR in at least 30% of the tumor samples from the corresponding grade, when compared to an average of the normal neural tissue expression. The results show that the  in silico  selection using SAGE profiles revealed tumor-associated genes that can be found in a different set of primary tumors implying a possible role for these genes in tumor development and increasing their value as putative therapeutic targets. The limited availability of high quality antibodies for the identified tumor-associated genes (Table  2 ) narrowed our study at the protein level to those genes that had previously been implicated in astrocytic tumors. Monoclonal antibodies for  TOP2A  and  AQ1  were used to analyze the expression at the protein level in individual glioblastoma sections and in a tissue micro-array. Strong nuclear staining in 5 of 8 individual glioblastomas tested was found for TOP2A (Figure  1A,1C  and  1D ). The GBMs showed intensely staining cells with the percentage of positive cells varying between 3 and 10%. Similar results were obtained when staining a tissue micro-array containing a different set of 20 GBMs. In six cases we found 5 to 10% of the cells expressing TOP2A. The percentage of strong positive cells among the 20 anaplastic astrocytomas present on the tissue micro array was lower and estimated to be 2 – 3% in 10 cases. Normal neural brain tissues like cortex, white matter, spinal cord and hippocampus where negative for TOP2A (Figure  1B ). Cytoplasmatic staining was observed when anti-aquaporin 1 monoclonal antibodies were used. Three of 5 individual GBM sections where positive, with examples shown in Figures  1F  and  1G . These results emphasize that the high transcript levels of TOP2A and Aquaporin 1 correlate well with a high protein level in a third independent set of tumors, implying that this might also be the case for the other identified tumor-associated genes as listed in Table  2  and Table  3 . Potential therapeutic targets in astrocytic tumors Encouraged by the confirmation of our initial  in silico  analysis for tumor-associated genes we formulated a slightly simplified approach to find cell surface, extra-cellular matrix and cell adhesion related genes. We selected for transcript tags with at least a ten fold over-expression when compared to the average expression level in normal neural brain tissues. Next we applied a filter that would include only those tags with an average expression of at least 5 or 10 counts per 100,000 tags in 30% or more of the tumors, respectively for anaplastic astrocytomas and glioblastomas. The generated lists of transcript tags were mapped to the corresponding gene using SAGE Genie, where after the gene ontology information (if available) was used as a final filter to identify membrane, cell surface and cell adhesion related genes (Table  3 ). Interestingly, almost 50% of the genes identified as highly expressed in both tumor types have not previously been implicated in astrocytomas and are potential new therapeutic targets. Intracellular proteins that contribute to the fusion of the vesicles with the plasma membrane during exocytosis include synaptosomal protein and vesicle-associated membrane proteins (VAMP). Both anaplastic astrocytomas and glioblastomas show high expression of VAPB and anaplastic astrocytomas express caveolin1 (Table  3 ). It has been shown that caveolae require intact VAMP for targeted transport in endothelial cells. Caveolae and associated proteins might be targeted in cancer as recently suggested [ 16 ]. One of the other genes expressed in common between the three astrocytomas is chitinase 3-like 2 ( CHI3L2 ) or  YKL-39  (Table  2 ).  CHI3L2  is a chondrocyte growth related gene and is an antigen found in rheumatoid arthritis [ 17 ] and osteoarthritis and a possible immunotherapy target. Another commonly over-expressed gene is  Neuromedin B . This neuropeptide has been implicated as an autocrine growth factor in lung cancer cells [ 18 ] that binds to a G protein-coupled receptor on the cell surface and might have a similar role in astrocytic tumors. It is tempting to speculate that a specific neuropeptide antagonist or neutralizing antibodies might reduce astrocytoma growth. Neuromedin B had previously been described as a GBM marker [ 9 ], and was included along with  ABCC3  in the real-time PCR analysis as a positive control. Another relatively unknown gene is the recently characterized  SMOC-1  [ 19 ], which was identified as a grade II and III tumor-associated gene (Table  2 ). This gene is related to SPARC/osteonectin, which was reported to participate in angiogenesis and tumor formation of human melanomas. Another extra cellular gene, Matrix Gla protein [ 20 ] had increased expression levels in higher-grade astrocytomas. MGP helps regulate the calcification of the extra cellular matrix [ 20 ]. Aquaporin 1 is an integral membrane protein important in the regulation of water transport in various epithelial and endothelial cell types [ 21 ]. The over-expression of  AQP1  in human brain tumors was described in a limited array study of 4 Glioblastomas [ 11 ] and it has been suggested that the protein might play a role in brain tumor edema in a similar way as the closely related aquaporin-4 [ 22 ]. Although the specific role of AQP1 in brain tumors is still unknown, our demonstration that AQP1 is consistently expressed in GBM may prompt other studies. Thymidilate synthetase and Topoisomarese 2A were over-expressed in glioblastoma as well as in our previous study of medulloblastoma [ 1 ]. Considering the role of Top2A as a molecular target of various anticancer drugs, and its identification as a survival marker in astrocytomas [ 23 ], its over-expression at the protein level in multiple brain tumors and the development of TOP2A inhibitors [ 24 ] makes the molecular targeting of TOP2A worthy of further investigation. In summary we have identified a number of new tumor-associated genes for three different grades of astrocytic tumors, and helped re-confirm in a larger set of samples several previously known astrocytoma genes. Despite the high heterogeneity among gliomas, a small set of genes is consistently observed at high levels in more than a third of each grade of astrocytoma studied. Many other cell surface, extra-cellular matrix or cell adhesion genes have been identify as potential targets for cancer therapy in astrocytic tumors. Although the therapeutic value of these markers is speculative at this point, by integrating this data onto the commonly used gene expression resource,  SAGE Genie , this data can be used as a standard to determine gene expression in astrocytomas. Further evaluation by  in vitro  and  in vivo  studies will be necessary to establish the role of these over-expressed genes in brain tumor development and progression.
Results and Discussion
SAGE gene expression profiles, selection and confirmation of tumor-associated genes This report describes the comprehensive generation of expression profiles of three astrocytic tumor grades based on Serial Analysis of Gene Expression (SAGE). The main goals were to identify genes not expressed in normal brain tissue and genes highly expressed in the more aggressive astrocytomas encoding cell surface or extra-cellular matrix related proteins that could be of potential therapeutic interest. We generated SAGE profiles on 8 infiltrating astrocytomas, 10 anaplastic astrocytomas and 7 glioblastoma samples. Combined with two glioblastoma profiles [ 8 ], previously deposited on SAGE Genie, this study analyzed 2,734,106 astrocytoma SAGE tags. On average we could distinguish over 27,000 unique tags in each tumor grade after excluding those with single counts (Table  1 ). Complete expression profiles and library information are posted for the scientific community at the Cancer Genome Anatomy Project (CGAP) SAGE Genie website  , where the libraries can be downloaded or viewed online using SAGE Genie tools [ 2 ]. In order to identify tumor-associated genes, we sought highly expressed transcripts in each grade of astrocytoma that were not expressed in 7 normal brain tissues. This also helped control for contaminating normal cells within the tumor sample. Tags expressed in a normal leukocyte SAGE library were also included in the analysis so they could be subtracted to reduce the chances of identifying transcripts from white blood cells that frequently infiltrate these tumors. Initially we selected for tags with an average expression of at least 3 per 100,000 tags. Subsequently, we selected for tags expressed at less than 2 counts per 100,000 in each of the 8 normal libraries (7 normal neural tissues & one leukocyte), reducing the number of tags to less than 100 per tumor type (Table  1 ). We further narrowed down the list of candidates by including only those tag sequences that could be matched with a full-length cDNA sequence. From these lists of genes we selected respectively 8, 16 and 10 genes for real-time PCR analysis in an independent set of 14 to 17 grade II, grade III and grade IV primary tumors. Only 6 genes could not be confirmed by real-time PCR analysis, 3 of 8 (grade II selection) and 3 of 16 (grade III selection). Another 8 genes were confirmed in only 20 to 25 % of the tumors. Table  2  lists those genes with a 5-fold or more over-expression by real time PCR in at least 30% of the tumor samples from the corresponding grade, when compared to an average of the normal neural tissue expression. The results show that the  in silico  selection using SAGE profiles revealed tumor-associated genes that can be found in a different set of primary tumors implying a possible role for these genes in tumor development and increasing their value as putative therapeutic targets. The limited availability of high quality antibodies for the identified tumor-associated genes (Table  2 ) narrowed our study at the protein level to those genes that had previously been implicated in astrocytic tumors. Monoclonal antibodies for  TOP2A  and  AQ1  were used to analyze the expression at the protein level in individual glioblastoma sections and in a tissue micro-array. Strong nuclear staining in 5 of 8 individual glioblastomas tested was found for TOP2A (Figure  1A,1C  and  1D ). The GBMs showed intensely staining cells with the percentage of positive cells varying between 3 and 10%. Similar results were obtained when staining a tissue micro-array containing a different set of 20 GBMs. In six cases we found 5 to 10% of the cells expressing TOP2A. The percentage of strong positive cells among the 20 anaplastic astrocytomas present on the tissue micro array was lower and estimated to be 2 – 3% in 10 cases. Normal neural brain tissues like cortex, white matter, spinal cord and hippocampus where negative for TOP2A (Figure  1B ). Cytoplasmatic staining was observed when anti-aquaporin 1 monoclonal antibodies were used. Three of 5 individual GBM sections where positive, with examples shown in Figures  1F  and  1G . These results emphasize that the high transcript levels of TOP2A and Aquaporin 1 correlate well with a high protein level in a third independent set of tumors, implying that this might also be the case for the other identified tumor-associated genes as listed in Table  2  and Table  3 .
SAGE gene expression profiles, selection and confirmation of tumor-associated genes
This report describes the comprehensive generation of expression profiles of three astrocytic tumor grades based on Serial Analysis of Gene Expression (SAGE). The main goals were to identify genes not expressed in normal brain tissue and genes highly expressed in the more aggressive astrocytomas encoding cell surface or extra-cellular matrix related proteins that could be of potential therapeutic interest. We generated SAGE profiles on 8 infiltrating astrocytomas, 10 anaplastic astrocytomas and 7 glioblastoma samples. Combined with two glioblastoma profiles [ 8 ], previously deposited on SAGE Genie, this study analyzed 2,734,106 astrocytoma SAGE tags. On average we could distinguish over 27,000 unique tags in each tumor grade after excluding those with single counts (Table  1 ). Complete expression profiles and library information are posted for the scientific community at the Cancer Genome Anatomy Project (CGAP) SAGE Genie website  , where the libraries can be downloaded or viewed online using SAGE Genie tools [ 2 ].In order to identify tumor-associated genes, we sought highly expressed transcripts in each grade of astrocytoma that were not expressed in 7 normal brain tissues. This also helped control for contaminating normal cells within the tumor sample. Tags expressed in a normal leukocyte SAGE library were also included in the analysis so they could be subtracted to reduce the chances of identifying transcripts from white blood cells that frequently infiltrate these tumors. Initially we selected for tags with an average expression of at least 3 per 100,000 tags. Subsequently, we selected for tags expressed at less than 2 counts per 100,000 in each of the 8 normal libraries (7 normal neural tissues & one leukocyte), reducing the number of tags to less than 100 per tumor type (Table  1 ). We further narrowed down the list of candidates by including only those tag sequences that could be matched with a full-length cDNA sequence. From these lists of genes we selected respectively 8, 16 and 10 genes for real-time PCR analysis in an independent set of 14 to 17 grade II, grade III and grade IV primary tumors. Only 6 genes could not be confirmed by real-time PCR analysis, 3 of 8 (grade II selection) and 3 of 16 (grade III selection). Another 8 genes were confirmed in only 20 to 25 % of the tumors. Table  2  lists those genes with a 5-fold or more over-expression by real time PCR in at least 30% of the tumor samples from the corresponding grade, when compared to an average of the normal neural tissue expression. The results show that the  in silico  selection using SAGE profiles revealed tumor-associated genes that can be found in a different set of primary tumors implying a possible role for these genes in tumor development and increasing their value as putative therapeutic targets.in silicoThe limited availability of high quality antibodies for the identified tumor-associated genes (Table  2 ) narrowed our study at the protein level to those genes that had previously been implicated in astrocytic tumors. Monoclonal antibodies for  TOP2A  and  AQ1  were used to analyze the expression at the protein level in individual glioblastoma sections and in a tissue micro-array. Strong nuclear staining in 5 of 8 individual glioblastomas tested was found for TOP2A (Figure  1A,1C  and  1D ). The GBMs showed intensely staining cells with the percentage of positive cells varying between 3 and 10%. Similar results were obtained when staining a tissue micro-array containing a different set of 20 GBMs. In six cases we found 5 to 10% of the cells expressing TOP2A. The percentage of strong positive cells among the 20 anaplastic astrocytomas present on the tissue micro array was lower and estimated to be 2 – 3% in 10 cases. Normal neural brain tissues like cortex, white matter, spinal cord and hippocampus where negative for TOP2A (Figure  1B ). Cytoplasmatic staining was observed when anti-aquaporin 1 monoclonal antibodies were used. Three of 5 individual GBM sections where positive, with examples shown in Figures  1F  and  1G . These results emphasize that the high transcript levels of TOP2A and Aquaporin 1 correlate well with a high protein level in a third independent set of tumors, implying that this might also be the case for the other identified tumor-associated genes as listed in Table  2  and Table  3 .TOP2AAQ1Potential therapeutic targets in astrocytic tumors Encouraged by the confirmation of our initial  in silico  analysis for tumor-associated genes we formulated a slightly simplified approach to find cell surface, extra-cellular matrix and cell adhesion related genes. We selected for transcript tags with at least a ten fold over-expression when compared to the average expression level in normal neural brain tissues. Next we applied a filter that would include only those tags with an average expression of at least 5 or 10 counts per 100,000 tags in 30% or more of the tumors, respectively for anaplastic astrocytomas and glioblastomas. The generated lists of transcript tags were mapped to the corresponding gene using SAGE Genie, where after the gene ontology information (if available) was used as a final filter to identify membrane, cell surface and cell adhesion related genes (Table  3 ). Interestingly, almost 50% of the genes identified as highly expressed in both tumor types have not previously been implicated in astrocytomas and are potential new therapeutic targets. Intracellular proteins that contribute to the fusion of the vesicles with the plasma membrane during exocytosis include synaptosomal protein and vesicle-associated membrane proteins (VAMP). Both anaplastic astrocytomas and glioblastomas show high expression of VAPB and anaplastic astrocytomas express caveolin1 (Table  3 ). It has been shown that caveolae require intact VAMP for targeted transport in endothelial cells. Caveolae and associated proteins might be targeted in cancer as recently suggested [ 16 ]. One of the other genes expressed in common between the three astrocytomas is chitinase 3-like 2 ( CHI3L2 ) or  YKL-39  (Table  2 ).  CHI3L2  is a chondrocyte growth related gene and is an antigen found in rheumatoid arthritis [ 17 ] and osteoarthritis and a possible immunotherapy target. Another commonly over-expressed gene is  Neuromedin B . This neuropeptide has been implicated as an autocrine growth factor in lung cancer cells [ 18 ] that binds to a G protein-coupled receptor on the cell surface and might have a similar role in astrocytic tumors. It is tempting to speculate that a specific neuropeptide antagonist or neutralizing antibodies might reduce astrocytoma growth. Neuromedin B had previously been described as a GBM marker [ 9 ], and was included along with  ABCC3  in the real-time PCR analysis as a positive control. Another relatively unknown gene is the recently characterized  SMOC-1  [ 19 ], which was identified as a grade II and III tumor-associated gene (Table  2 ). This gene is related to SPARC/osteonectin, which was reported to participate in angiogenesis and tumor formation of human melanomas. Another extra cellular gene, Matrix Gla protein [ 20 ] had increased expression levels in higher-grade astrocytomas. MGP helps regulate the calcification of the extra cellular matrix [ 20 ]. Aquaporin 1 is an integral membrane protein important in the regulation of water transport in various epithelial and endothelial cell types [ 21 ]. The over-expression of  AQP1  in human brain tumors was described in a limited array study of 4 Glioblastomas [ 11 ] and it has been suggested that the protein might play a role in brain tumor edema in a similar way as the closely related aquaporin-4 [ 22 ]. Although the specific role of AQP1 in brain tumors is still unknown, our demonstration that AQP1 is consistently expressed in GBM may prompt other studies. Thymidilate synthetase and Topoisomarese 2A were over-expressed in glioblastoma as well as in our previous study of medulloblastoma [ 1 ]. Considering the role of Top2A as a molecular target of various anticancer drugs, and its identification as a survival marker in astrocytomas [ 23 ], its over-expression at the protein level in multiple brain tumors and the development of TOP2A inhibitors [ 24 ] makes the molecular targeting of TOP2A worthy of further investigation. In summary we have identified a number of new tumor-associated genes for three different grades of astrocytic tumors, and helped re-confirm in a larger set of samples several previously known astrocytoma genes. Despite the high heterogeneity among gliomas, a small set of genes is consistently observed at high levels in more than a third of each grade of astrocytoma studied. Many other cell surface, extra-cellular matrix or cell adhesion genes have been identify as potential targets for cancer therapy in astrocytic tumors. Although the therapeutic value of these markers is speculative at this point, by integrating this data onto the commonly used gene expression resource,  SAGE Genie , this data can be used as a standard to determine gene expression in astrocytomas. Further evaluation by  in vitro  and  in vivo  studies will be necessary to establish the role of these over-expressed genes in brain tumor development and progression.
Potential therapeutic targets in astrocytic tumors
Encouraged by the confirmation of our initial  in silico  analysis for tumor-associated genes we formulated a slightly simplified approach to find cell surface, extra-cellular matrix and cell adhesion related genes. We selected for transcript tags with at least a ten fold over-expression when compared to the average expression level in normal neural brain tissues. Next we applied a filter that would include only those tags with an average expression of at least 5 or 10 counts per 100,000 tags in 30% or more of the tumors, respectively for anaplastic astrocytomas and glioblastomas. The generated lists of transcript tags were mapped to the corresponding gene using SAGE Genie, where after the gene ontology information (if available) was used as a final filter to identify membrane, cell surface and cell adhesion related genes (Table  3 ). Interestingly, almost 50% of the genes identified as highly expressed in both tumor types have not previously been implicated in astrocytomas and are potential new therapeutic targets.in silicoIntracellular proteins that contribute to the fusion of the vesicles with the plasma membrane during exocytosis include synaptosomal protein and vesicle-associated membrane proteins (VAMP). Both anaplastic astrocytomas and glioblastomas show high expression of VAPB and anaplastic astrocytomas express caveolin1 (Table  3 ). It has been shown that caveolae require intact VAMP for targeted transport in endothelial cells. Caveolae and associated proteins might be targeted in cancer as recently suggested [ 16 ].One of the other genes expressed in common between the three astrocytomas is chitinase 3-like 2 ( CHI3L2 ) or  YKL-39  (Table  2 ).  CHI3L2  is a chondrocyte growth related gene and is an antigen found in rheumatoid arthritis [ 17 ] and osteoarthritis and a possible immunotherapy target. Another commonly over-expressed gene is  Neuromedin B . This neuropeptide has been implicated as an autocrine growth factor in lung cancer cells [ 18 ] that binds to a G protein-coupled receptor on the cell surface and might have a similar role in astrocytic tumors. It is tempting to speculate that a specific neuropeptide antagonist or neutralizing antibodies might reduce astrocytoma growth. Neuromedin B had previously been described as a GBM marker [ 9 ], and was included along with  ABCC3  in the real-time PCR analysis as a positive control.CHI3L2YKL-39CHI3L2Neuromedin BABCC3Another relatively unknown gene is the recently characterized  SMOC-1  [ 19 ], which was identified as a grade II and III tumor-associated gene (Table  2 ). This gene is related to SPARC/osteonectin, which was reported to participate in angiogenesis and tumor formation of human melanomas. Another extra cellular gene, Matrix Gla protein [ 20 ] had increased expression levels in higher-grade astrocytomas. MGP helps regulate the calcification of the extra cellular matrix [ 20 ].SMOC-1Aquaporin 1 is an integral membrane protein important in the regulation of water transport in various epithelial and endothelial cell types [ 21 ]. The over-expression of  AQP1  in human brain tumors was described in a limited array study of 4 Glioblastomas [ 11 ] and it has been suggested that the protein might play a role in brain tumor edema in a similar way as the closely related aquaporin-4 [ 22 ]. Although the specific role of AQP1 in brain tumors is still unknown, our demonstration that AQP1 is consistently expressed in GBM may prompt other studies.AQP1Thymidilate synthetase and Topoisomarese 2A were over-expressed in glioblastoma as well as in our previous study of medulloblastoma [ 1 ]. Considering the role of Top2A as a molecular target of various anticancer drugs, and its identification as a survival marker in astrocytomas [ 23 ], its over-expression at the protein level in multiple brain tumors and the development of TOP2A inhibitors [ 24 ] makes the molecular targeting of TOP2A worthy of further investigation.In summary we have identified a number of new tumor-associated genes for three different grades of astrocytic tumors, and helped re-confirm in a larger set of samples several previously known astrocytoma genes. Despite the high heterogeneity among gliomas, a small set of genes is consistently observed at high levels in more than a third of each grade of astrocytoma studied. Many other cell surface, extra-cellular matrix or cell adhesion genes have been identify as potential targets for cancer therapy in astrocytic tumors. Although the therapeutic value of these markers is speculative at this point, by integrating this data onto the commonly used gene expression resource,  SAGE Genie , this data can be used as a standard to determine gene expression in astrocytomas. Further evaluation by  in vitro  and  in vivo  studies will be necessary to establish the role of these over-expressed genes in brain tumor development and progression.SAGE Geniein vitroin vivoAuthors' contributions KB performed computational analyses, generated experimental data, participated in the design of the study and drafted the manuscript. JBE generated experimental data. CBE and GJR participated in the study design and manuscript editing. All authors read and approved the final manuscript.
Authors' contributions
KB performed computational analyses, generated experimental data, participated in the design of the study and drafted the manuscript. JBE generated experimental data. CBE and GJR participated in the study design and manuscript editing. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: