Surgical extent impacts the value of the established prognosticators in glioblastoma patients: a prospective translational study in Asia

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1 Page 1 of 11 Surgical extent impacts the value of the established prognosticators in glioblastoma patients: a prospective translational study in Asia Y Wang 1,2, S Li 3, Z Zhang 1, X Chen 4, G You 1, P Yang 1, W Yan 1, Z Bao 1, K Yao 5, L Wang 1, M Li 2 *, T Jiang 1 * Abstract Background While multimodal treatments have survival benefits in patients with glioblastoma, tumour volume resection still remains the most effective treatment. Methods In this prospective translational study, we analysed the clinical values of the established survival predictors in the context of extensive tumour resection (at least near-total resection) in 234 newly diagnosed glioblastoma patients. Common survival factors such as adjuvant therapy modality, O 6 -methylguanine DNA methyltransferase (MGMT) and isocitrate dehydrogenase 1 (IDH1) were analysed. Results The more extensive resection resulted in a favourable outcome, especially the median progression-free survival up to 11.1 months. Age at diagnosis, the initial alkylating radiochemotherapy and MGMT promoter *Corresponding authors min.li@uth.tmc.edu; taojiang1964@ yahoo.com.cn 1 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing , China 2 The Vivian L. Smith Department of Neurosurgery, University of Texas Medical School at Houston, Houston, TX, USA 3 Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing , China 4 Department of Neuroimaging, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 5 Department of Pathology, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing, China status were correlated with survival in univariate analysis, but their independent predictive values were lost upon multivariate analysis. Multivariate analysis identified the only independent prognosticators for a longer overall survival were gross total resection (relative risk [RR] = 0.36; P = 0.003) and higher Karnofsky Performance Status score (RR = 0.41; P = 0.008), and gross-total resection (RR = 0.56; P = 0.050), higher Karnofsky Performance Status score (RR = 0.52; P = 0.028) and IDH1 mutation (RR = 0.42; P = 0.011) for a favourable progression-free survival. Interestingly, in the subgroup of patients receiving gross-total resection, neither MGMT nor IDH1 effectively predicted overall survival or progression-free survival; and also radiotherapy alone did not significantly improve the outcome though alkylating radiochemotherapy showed a strong survival advantage. Further analysis showed an additive effect of surgical extent and molecular predictor IDH1 mutation. Conclusion Surgical extent most significantly impacts the value of molecular and clinical predictors, and therefore should be taken into full account when assessing a new therapy or stratification variable in glioblastoma multiformes. Introduction Glioblastoma multiforme (GBM) is the most common malignant brain tumour and remains a major therapeutic challenge. The survival benefits associated with the adjuvant treatment modalities and the values of clinical and genetic markers are under wide investigation. To date, patient s age at diagnosis and preoperative Karnofsky Performance Status (KPS) score have been well documented as predictors of survival 1. In several large-scale clinical trials, three molecular markers have been confirmed to have any clinical significance for the prognosis of malignant gliomas 2 : O 6 -methylguanine DNA methyltransferase (MGMT) promoter hypermethylation has been associated with prolonged survival in patients treated with alkylating agents such as nitrosoureas or temozolomide 3 6 ; loss of heterozygosity on chromosome arms 1p and 19q predicted a favourable outcome in patients with oligodendroglial gliomas treated with chemotherapy and/or radiotherapy 7,8 ; and also isocitrate dehydrogenase 1 (IDH1) mutation has suggested to be a potent predictor for favourable survival in gliomas in the recent striking studies Most noteworthy, alkylating radiochemotherapy after surgical resection predicted unequivocally longer overall survival (OS) than radiotherapy alone in the Stupp et al. s phase III clinical trial 12 ; since then, the protocol has become a standard treatment for patients with newly diagnosed GBM 13. Tumour volume resection, however, currently remains the most effective treatment for patients with GBM. Mounting evidence suggests that more extensive surgical resection is strongly associated with longer life expectancy for high-grade gliomas The quality of surgical resection should thus be taken emphatically into account when assessing the clinical values of stratification factors and the benefit of adjuvant therapies. However, in the context of more extensive tumour resection, the

2 Page 2 of 11 clinical values of the popular adjuvant therapy regimens and the common prognosticators remain unknown. Advances in brain-mapping technologies such as preoperative functional magnetic resonance imaging (fmri), awake mapping, intra-operative ultrasound and neuronavigation made the maximal tumour bulk resection with preservation of the key eloquent area become attainable. In the present study, we evaluated the clinical values of common survivalrelated factors in a prospective cohort of 234 patients with newly diagnosed GBM who underwent at least neartotal resection (NTR). Patients and methods Patients A prospective cohort of patients with newly diagnosed and histologically confirmed GBM were recruited at the Glioma Center of Beijing Tiantan Hospital from March 2005 to March The surgical aim was maximal tumour bulk resection with preservation of the key eloquent area (motor and language area). Preoperative fmri and awake mapping (intra-operative cortical electrical stimulation technique) were used for tumours involving the eloquent area 17. Surgical interventions and subsequent medical management were performed by the same group of surgeons following identical procedures. Extent of resection (EOR) was estimated by post-operative gadolinium-enhanced MRI (Siemens, 3.0 T) within 3 days after surgery. Based on the MRI results, gross-total resection (GTR) was defined as no residual enhancement (resection margins 1 2 cm away from tumour border), NTR as having thin rim enhancement of the resection cavity only (resection margins at the level of gadoliniumenhanced border), and sub-total resection as having residual nodular enhancement. The EOR was independently determined by two experienced radiologists, who were blinded to the clinical data of the patients. In case of a discrepancy, the two observers simultaneously reviewed the images to achieve a consensus. A total of 256 patients who underwent at least NTR were admitted into this trial. Eligible patients were aged years, with a preoperative KPS 50, and with adequate hematologic, renal and hepatic function (absolute neutrophil count /L; platelet count /L; serum creatinine level 1.5 times the upper limit of normal and liver function values <3 times the upper limit of the normal). All patients provided written informed consent, and the study was approved by the Research Ethics Board of Beijing Tiantan Hospital. Radiotherapy at a total dose of 60 Gy was administered within 4 weeks after surgery (2 Gy each dose, with 5 fractions administered per week). The tumour volume initially treated included the contrast-enhanced lesion and the surrounding oedema identified by pre-operative MRI plus a 2 3 cm margin. After 46 Gy had been administered, treatment was confined to the contrast-enhanced lesion only. First-line adjuvant chemotherapy consisted of a standard temozolomide-based scheme 12 or nitrosourea-based scheme 18. A total of six cycles were administered if no disease progression occurred and no irreversible haematological toxic effects were observed. Treatment response was assessed by regular enhanced MRI scans at 3-month intervals, and progression was defined as the appearance of a new lesion or an increase in tumour size of 25% 19. Failure to return for evaluation due to death or a deteriorating condition was considered to represent progression. The clinical end-points used to measure clinical outcome were OS and progressionfree survival (PFS). OS was calculated from the day of first surgery until death or end of follow-up. PFS was calculated from the day of first surgery until tumour progression, death or end of follow-up. Pyrosequencing for IDH1 mutation and MGMT promoter methylation Specimens with an estimated tumour-cell content of 80% were selected. For IDH1 mutation analysis, genomic DNA was isolated from frozen tumour tissues using the QIAamp DNA Mini Kit (Qiagen). The genomic region spanning wild-type R132 of IDH1 was analysed by pyrophosphate sequencing using the following primers: 5 -GCTTGTGAGTG- GATGGGTAAAAC-3 and 5 -biotin- TTGCCAACATGACTTACTT GATC-3. For MGMT promoter methylation analysis, bisulphite modification of the DNA was performed using the EpiTect Kit (Qiagen). The primers used were 5 -GTTTYGGATATGTTGGGATA-3 and reverse 5 -biotin-acccaaacactcac- CAAATC-3. Pyrosequencing analysis of IDH1 mutation and MGMT promoter methylation was carried out by Gene Tech Company Limited (Shanghai, China). The methylation values obtained were averaged across the seven tested CpG loci within the MGMT promoter. GBM samples were considered MGMT promoter methylation-positive if they had an average methylation of >10% 11. Immunohistochemistry for MGMT expression Immunoperoxidase staining for MGMT (Santa Cruz Biotechnology, Santa Cruz, CA) was performed on formalin-fixed, paraffin-embedded tissue sections according to the manufacturer s instructions. Each stained slide was jointly scored by two independent pathologists blinded to the clinical information. In the event of a discrepancy, the two observers reviewed the slides simultaneously to achieve a consensus. High expression of MGMT was defined as >30% positively stained cells in the tumour. Fluorescence in situ hybridization for 1p/19q abnormalities Representative tumour areas were marked on haematoxylin and eosinstained sections. The corresponding

3 Page 3 of 11 areas were identified on the paraffin blocks, and new 4-µm sections were prepared. The material was deparaffinized with xylene, incubated with 0.3% pepsin in 10 mm HCl at 37 C for 10 minutes, and then denatured at 85 C for 10 minutes. Dual-colour fluorescence in situ hybridizations were performed using LSI probe sets 1p36/1q25 and 19q13/19p13 (spectrum orange-labelled 1p36 and 19q13 probes; spectrum green-labelled 1q25 and 19p13 probes; Vysis, Downers Grove, IL) and assessed in at least 200 non-overlapping nuclei with intact morphology. Statistical analysis All statistical analyses were performed using SPSS 13.0 for Windows (Chicago, IL). Correlations between two variables were analyzed by t tests, χ 2 tests, or Spearman s correlation. Survival curves were estimated using the Kaplan Meier method, and statistical differences were evaluated using the two-sided log-rank test. Cox proportional hazard regression analyses were applied to all patients who received either radiotherapy alone or radiotherapy plus chemotherapy as their first-line therapy. A value of P < 0.05 (two-sided) was considered to be statistically significant. Results Clinical data Patient demographics and baseline characteristics are summarized in Table 1. In all, 22 of the 256 cases were excluded from survival analysis because of loss to follow-up (n = 16), death during the peri-operative period (n = 3) and death due to non-gliomarelated causes (n = 3). A total 234 (91.4%) patients were included for survival analysis. Awake mapping was performed in 19 patients harbouring tumours in the key eloquent area to avoid neurological deficits. All the patients were Asian (Chinese Han people accounted for 96.2%), including 146 men and 88 women. The median age was 46.5 years (range = Table 1 Patient demographics and baseline characteristics Characteristic All patients (n = 256) years) and the median KPS score was 80 (range = ). A total of 194 patients received radiotherapy, 145 alkylating chemotherapy, and 135 received both as the initial treatment. A total of 93 patients were estimated to be treated with GTR surgery based on the MRI results after surgery, and 141 Patients used for survival analysis (n = 234) Age at diagnosis (years) Median (IQR) 47.0 (37 55) 46.5 (37 55) Age category (11.7%) 27 (11.5%) (49.2%) 113 (48.3%) > (39.1%) 94 (40.2%) Gender Male 161 (62.9%) 146 (62.4%) Female 95 (37.1%) 88 (37.6%) Race Chinese Han 247 (96.5%) 225 (96.2%) Others 9 (3.5%) 9 (3.8%) Preoperative KPS score Median (range) 80 (50 100) 80 (50 100) (61.7%) 143 (61.1%) <80 98 (38.3%) 91 (38.9%) Surgery a Gross-total resection 106 (41.4%) 93 (39.7%) Near-total resection 150 (58.6%) 141 (60.3%) First-line therapy RT plus TMZ 34 (14.5%) RT plus ACNU 101 (43.2%) RT alone 59 (24.8%) TMZ alone 1 (0.4%) ACNU alone 9 (3.8%) No adjuvant therapy 19 (8.0%) Unknown 11 (4.6%) Salvage surgery Yes 17 (7.3%) No 217 (92.7%) IQR, inter-quartile range; KPS, Karnofsky Performance Status; RT, radiotherapy; TMZ, temozolomide; ACNU, nimustine. a Based on magnetic resonance imaging results obtained within 72 hours after surgery, gross-total resection was defined as no residual enhancement, and near-total resection as having thin rim enhancement of the resection cavity only.

4 Page 4 of 11 patients were treated with NTR surgery. The clinical and genetic parameters in the two surgical groups were well balanced except for preoperative KPS score (Supplementary Table S1). At a median follow-up of 48.5 months (range = months), the median PFS was 11.1 months (95% confidence interval = months), and the PFS rates were 74% at 6 months and 44% at 12 months. The median OS was 14.1 months (95% confidence interval = months), with 1-, 2- and 5-year OS rates of 64%, 27% and 9%, respectively (Table 2). When compared with previously published reports 5,20, these relatively favourable results (especially for PFS) may reflect the fact that the present study cohort included only patients who underwent at least NTR. Molecular genetic markers IDH1 status was assessed by pyrophosphate sequencing in 131 tumour specimens from which frozen GBM tissues could be obtained. Mutations were detected in 29 tumours (22.1%), and all mutations were located at amino acid residue 132. The mutations in 27 of the 29 specimens (93.1%) were G395A transition (Arg His), followed by C394A transversion (Arg Ser; 6.9%). The ages of patients with IDH1 mutations were significantly lower than those of patients with intact IDH1 (median, 41 vs. 51 years; P = 0.04, t test). MGMT promoter methylation was detected in 33 of 101 tested cases (32.7%), and low MGMT protein expression was found in 111 of 232 cases (47.8%) (Table 3). The concordant MGMT methylation status and protein expression results were observed in 63 of 101 tumour specimens (62.4%; P = 0.034, Spearman s correlation). Fourteen tumours with MGMT methylation had high MGMT protein expression and 24 tumours with unmethylated MGMT had low MGMT protein expression. Chromosomal status assessment was limited to 49 tumour specimens because only one patient (<2%) had a combined 1p/19q deletion, which was too rare to be of prognostic value for GBM patients. Univariate analysis To analyse the clinical values of the initial adjuvant treatments in the context of at least NTR, patients were divided into three groups: chemotherapy plus radiotherapy, radiotherapy alone and no adjuvant therapy. Kaplan Meier estimates for PFS and OS are shown in Figure 1 and Table 3. Compared with surgical resection alone, radiotherapy significantly prolonged OS (P = 0.003) and tended to improve PFS (P = 0.096). The addition of alkylating agents to radiotherapy as a first-line therapy did not significantly improve PFS (P = 0.31) (Figure 1A), and only marginally improved OS (P = 0.10) (Figure 1B). MGMT promoter status showed a trend towards improved OS (P = 0.051) and PFS (P = 0.092), but there Table 2 Progression-free survival and overall survival in 234 patients with newly diagnosed glioblastoma Progression-free survival Overall survival Median (95% confidence interval) (days) 334 ( ) 423 ( ) at 6 months 74% 91% at 1 year 44% 64% at 2 years 18% 27% at 5 years 4% 9% was no significant association between its protein expression and either PFS or OS (Table 3). Further subgroup analysis showed that MGMT promoter methylation was significantly associated with a longer OS (26.3 vs months; P = 0.01) and PFS (17.7 vs months; P = 0.047) only in patients receiving alkylating chemotherapy (Figure 2A and B), but it did not translate into a significant prolongation of either PFS (P = 0.84) or OS (P = 0.86) in patients without alkylating chemotherapy (Figure 2C and D). Complete resection and IDH1 mutation were significantly correlated with improved PFS and OS (all Ps < 0.05) (Table 3). In view of GTR significantly associated with the favourable prognosis, we further analysed the subgroup of patients who received GTR surgery (93 patients; Figure 3). Interestingly, neither IDH1 nor MGMT effectively predicted either OS or PFS in the context of GTR. The relevant factors for survival were age, KPS score and adjuvant treatment modality (all Ps < 0.05). Notably, radiotherapy alone as first-line therapy did not significantly improve outcome, though alkylating radiochemotherapy showed a strong survival advantage. Multivariate analysis Factors with P values <0.1 in univariate analysis were introduced into the multivariate model, including age, KPS score, surgical resection, adjuvant therapy, IDH1 and MGMT. Backward stepwise variable selection was used to evaluate their independent prognostic values. Cox analyses were restricted to the two patient populations who received alkylating chemotherapy plus radiotherapy or radiotherapy alone as the initial treatment (194 patients; Table 4). Factors identified as independent prognostic factors for a longer OS were GTR (P = 0.003; relative risk [RR] = 0.36) and higher KPS score (P = 0.008; RR = 0.41). Prognostic factors for a favourable PFS were higher KPS score (P = 0.028; RR = 0.52), GTR (P = 0.050; RR = 0.56) and IDH1 mutation

5 Page 5 of 11 Table 3 Univariate analyses of potential prognostic survival factors (n = 234) PFS OS Parameter n (%) Median (95% CI) (days) P value a Median (95% CI) (days) P value a Age (years) (42.7) 289 ( ) ( ) < (57.3) 378 ( ) 514 ( ) Gender Male 146 (62.4) 328 ( ) ( ) Female 88 (37.6) 344 ( ) 422 ( ) Preoperative KPS score (61.1) 365 ( ) ( ) < (38.9) 258 ( ) 383 ( ) Surgery Gross-total resection 93 (39.7) 388 ( ) ( ) Near-total resection 141 (60.3) 292 ( ) 393 ( ) First-line therapy (n = 213) RT plus CT 135 (64.4) 344 ( ) ( ) RT alone 59 (27.7) 349 ( ) 401 ( ) No adjuvant therapy 19 (8.9) 162 (NR 347) 276 (70 482) Salvage surgery Yes 17 (7.3) 561 ( ) No 217 (92.7) 412 ( ) IDH1 (n = 131) Mutated 29 (22.1) 407 ( ) ( ) Wild-type 102 (77.9) 304 ( ) 393 ( ) MGMT promoter gene (n = 101) Methylated 33 (32.7) 353 (84 622) ( ) Unmethylated 68 (67.3) 314 ( ) 387 ( ) MGMT expression (n= 232) High 121 (51.7) 317 ( ) ( ) Low 111 (48.3) 348 ( ) 446 ( ) PFS, progression-free survival; OS, overall survival; CI, confidence interval; KPS, Karnofsky Performance Status; RT, radiotherapy; CT, chemotherapy; NR, not reached; IDH1, isocitrate dehydrogenase 1; MGMT, O 6 -methylguanine DNA methyltransferase. a Figures in boldface indicate statistically significant values (P <.05). (P = 0.011; RR = 0.42). Both age (P = 0.056) and IDH1 mutation (P = 0.062) were only marginally significant predictors of longer OS. However, MGMT promoter methylation and alkylating radiochemotherapy as initial therapy did not independently predict favourable OS or PFS. Additive effect on survival of surgical extent and molecular predictors Based on the observation that EOR and IDH1 gene status were both significantly associated with favourable outcome, we further divided the patients into four groups: group 1, GTR surgery and IDH1 mutation; group 2, NTR surgery and IDH1 mutation; group 3, GTR surgery and IDH1 wildtype; and group 4, NTR surgery and IDH1 wild-type (Table 5, Supplementary Figure S1). Survival analysis revealed that group 1 patients had significantly longer PFS and OS than group 2, 3 and 4 patients. Survival

6 Page 6 of 11 Figure 1: Kaplan Meier estimates of progression-free survival (PFS) (A) and overall survival (OS) (B) in the context of at least near-total resection, according to initial treatment with radiotherapy (RT) alone, RT plus chemotherapy (CT) combined, or no adjuvant therapy. times did not significantly different between group 2 and 3 patients, whereas group 4 patients showed significantly the poorer OS and PFS than the other three groups. Discussion This clinical translational study investigated the impact of the surgical extent on the clinical values of established prognostic markers in patients with newly diagnosed GBM in a large-sample single-institution series. The results demonstrated that the prognostic powers of the most common survival predictors, such as age at diagnosis, initial alkylating agent radiochemotherapy 5,12, IDH1 Figure 2: Sub-group analysis of the predictive and prognostic values of MGMT promoter status. MGMT methylation predicts survival in patients with alkylating chemotherapy (A, B) but not in patients without chemotherapy (C, D). mutation 9 11 and MGMT promoter methylation 4,5,7,16 were lost or reduced in patients undergoing a more extensive tumour resection. Also interestingly, in the subgroup of patients receiving GTR surgery, radiotherapy alone did not significantly improve outcome, though alkylating radiochemotherapy showed a strong survival advantage. These unusual results reveal that quality of surgical resection is a pivotal prerequisite when analysing the prognostic factors in patients with GBM. Despite recent advances in adjuvant treatments for GBM, the majority of large-sample studies have considered extensive surgical resection to be a significant independent predictor of favourable survival 14, The current study defined the EOR in detail based on postoperative MRI performed early after surgery. In line with the results of a study involving a large cohort of 1,215 malignant gliomas by McGirt et al. 21, we found that even among patients receiving at least NTR surgery, GTR remained a strong prognostic factor for improved survival in both univariate and multivariate analyses. While we noticed that the 16.1% patients in the GTR group reduced the post-operative KPS score (estimated at 2 weeks after operation), which is slightly higher than that in the NTR group (12.8%) (Supplementary Table S1). In fact, about half of them with the tumour involving the supplementary motor area, which surgical intervention resulted in immediate post-operative supplementary motor area syndrome (difficulty in performing voluntary motor acts in the contralateral limbs and aphasic symptoms [dominant hemisphere]) 25,26, and most of them would be recovered within 2 months. Most published studies that focus on survival-related factors in GBM, 4,5,15,16 including Stupp et al. s phase III clinical trial 12, contained high proportions of patients who had only undergone biopsies or partial resections. In the context of at least NTR, the current

7 Page 7 of 11 Figure 3: Clinical values of the popular molecular and clinical prognosticators in the subgroup of patients receiving gross total resection (GTR) surgery (n = 93). Neither IDH1 nor MGMT effectively predicts either overall survival (OS) or progression-free survival (PFS) in the context of GTR. The relevant factors for survival are age, Karnofsky Performance Status (KPS) score and adjuvant treatment modality. Notably, radiotherapy (RT) alone as first-line therapy does not show significantly improved outcome, though alkylating radiochemotherapy (RT + CT) shows a strong survival advantage. study found that the addition of an alkylating agent to radiotherapy as initial therapy was not associated with an independent survival benefit. Somewhat surprisingly, the prognostic power of age at diagnosis, a classic prognostic factor in GBM series 1, was also weakened in multivariate analysis. It is likely that this discrepancy can be accounted for by the fact that all the patients in the current series received at least NTR surgery. We therefore propose that more extensive resection should not be withheld from patients only on the basis of age. MGMT, IDH1 and chromosome arms 1p/19q are the three most common molecular markers in gliomas 2. Though loss of heterozygosity 1p/19q has been used as a prognostic marker in our institute 8, its clinical value in GBM is limited by its rarity (<2%). In agreement with the study by Criniere et al. 27, our current results showed that MGMT methylation only had predictive significance in patients who received alkylating chemotherapy, yet its value was lost in the final multivariate Cox model. So MGMT should thus be regarded cautiously as a predictive and/or prognostic marker of GBM, especially in the context of extensive surgical resection. MGMT protein expression and its gene methylation status were not always matched (inconsistency rate of 37.6%), and the latter was not linked with outcome, as reported previously 28. There could be several reasons for the discrepancy 29,30, suggesting the impracticability of MGMT evaluation by an immunohistochemical approach. In accordance with a previous report 10, IDH1 mutations were more likely to occur in younger

8 Page 8 of 11 Table 4 Multivariate analysis of progression-free survival and overall survival a PFS OS Variable HR 95% CI P value HR 95% CI P value Age 50 years NS Preoperative KPS score 80% Gross-total resection surgery RT plus CT (vs. RT alone) NS NS IDH1 mutation MGMT gene methylation NS NS PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; RT, radiotherapy; CT, chemotherapy; NS, not significant; KPS, Karnofsky Performance Status; IDH1, isocitrate dehydrogenase 1; MGMT, O 6 -methylguanine DNA methyltransferase. a n = 194 patients received either CT plus RT or RT alone. Backward stepwise elimination with a threshold P value.1 was used to select variables in the final model. patients and predicted a better clinical outcome in GBM. IDH1 mutations have recently been shown to be an early event in tumourigenesis, and are the best available genetic hallmark of secondary GBMs, which usually have a favourable prognosis 31. IDH1 mutations are thus valuable for detecting clinically silent secondary GBMs (without a history of lower grade glioma) among primary ones. However, it should be noted that the prognostic power of IDH1 was limited in patients undergoing GTR. Furthermore, our findings suggested an additive effect of surgical extent and IDH1 gene status, with those patients having IDH1 mutation and complete resection showing the best prognosis. Patients having one of them showed an intermediated prognosis, whereas patients having IDH1 wild-type and incomplete resection showed the worst outcome. These findings strongly suggest that more extensive tumour resection should be attempted in GBM patients, with the preservation of the Table 5 Additive effect on survival of surgical extent and IDH1 gene status (n = 131) Median OS (95% CI) (days) Median PFS (95% CI) (days) P value Group 1 GTR surgery and IDH1 mutation 605 ( ) 531 ( ) Group 2 NTR surgery and IDH1 mutation 518 ( ) 353 ( ) Group 3 GTR surgery and IDH1 wild-type 486 ( ) 396 ( ) Group 4 NTR surgery and IDH1 wild-type 363 ( ) 272 ( ) IDH1, isocitrate dehydrogenase 1; OS, overall survival; PFS, progression-free survival; CI, confidence interval; GTR, gross-total resection; NTR, near-total resection. key neurological function under the guidance of modern brain-mapping technologies. One possible limitation of this prospective study was that the threshold for high-quality surgical resection may have excluded some elderly GBM patients (age >65 years), most of whom only underwent biopsies or debulking surgery. Several recent studies have shown that elderly patients may demonstrate a distinctive prognostic pattern 32,33. Otherwise, inter-ethnic difference is a non-negligible factor. Studies in other types of tumours have indicated that ethnic differences attribute to genetic differences and have distinctive prognostic models All the patients in the current study were Asian (Han Chinese, 96%), whereas the previous studies were primarily in Caucasians 3 5,15,16, Therefore, these findings need to be further validated in Caucasian setting. In summary, this large-scale study investigated the clinical values of current prognostic factors in Asian GBM patients in the context of more extensive surgical resection. The results demonstrated that the prognostic powers of the common predictors may be limited or narrowed in patients undergoing at least NTR surgery. The

9 Page 9 of 11 quality of surgical resection strongly impacts the clinical value of prognostic markers in GBM, and should thus be taken into account when assessing potential stratification variables and the benefits of adjuvant therapeutic modalities in future trials. Abbreviations list EOR, extent of resection; GBM, glioblastoma multiforme; GTR, gross-total resection; IDH1, isocitrate dehydrogenase 1; KPS, Karnofsky Performance Status; MGMT, O 6 -methylguanine DNA methyltransferase; MRI, magnetic resonance imaging; NTR, near-total resection; OS, overall survival; PFS, progression-free survival. Acknowledgements We thank Yuling Yang for tissue sample collection and clinical data retrieval. This study was funded by the National High Technology Research and Development Program 2012AA02A508, International Science and Technology Cooperation Program 2012DFA30470, Biological Sciences Training Program D (T Jiang) and Dr. Marnie Rose Foundation (M Li). Supplementary material Table S1 Features of the patients undergoing near-total resection or gross-total resection Parameter NTR GTR P Value Number of patients Age (years) 50 (%) 55 (39.0) 42 (45.2) <50 (%) 86 (61.0) 51 (54.8) Gender Female (%) 52 (36.9) 36 (38.7) Pre-operative KPS score 80 (%) 78 (55.3) 65 (69.9) <80 (%) 63 (44.7) 28 (30.1) KPS score change at 2 weeks after operation 0 (%) 123 (87.2) 78 (83.9) <0 (%) 18 (12.8) 15 (16.1) First-line therapy (n = 213) RT plus CT (%) 79 (62.2) 56 (65.1) RT alone (%) 37 (29.1) 22 (25.6) No adjuvant therapy (%) 11 (8.7) 8 (9.3) IDH1 mutation (n = 131) Yes (%) 20 (26.7) 9 (16.7) MGMT promoter gene (n = 101) Methylated (%) 22 (34.9) 11 (30.6) MGMT expression (n = 232) Low expression (%) 72 (51.4) 39 (42.4) LOH 1p and 19q (n = 49) a Yes (%) 1 (3.6) 0 (0.0) NTR, near-total resection; GTR, gross-total resection; KPS, Karnofsky Performance Status; RT, radiotherapy; CT, chemotherapy; IDH1, isocitrate dehydrogenase 1; MGMT, O 6 -methylguanine DNA methyltransferase; LOH, loss of heterozygosity. a The chromosome status assessment was limited to 49 tumour specimens because the low incidence of 1p/19q co-deletion indicates that it is not a valued prognostic indicator for patients with glioblastoma multiforme.

10 Page 10 of 11 Figure S1: Survival stratification according to the surgical extent and IDH1 gene status. The patients having complete resection and IDH1 mutation show the best overall survival (A) and progression-free survival (B); in contrast, the patients having incomplete resection and IDH1 wild-type show the worst prognosis. The patients having one of them showed an intermediated prognosis. GTR, gross total resection; NTR, near-total resection. References 1. Siker ML, Wang M, Porter K, Nelson DF, Curran WJ, Michalski JM, et al. Age as an independent prognostic factor in patients with glioblastoma: a Radiation Therapy Oncology Group and American College of Surgeons National Cancer Data Base comparison. J Neurooncol Aug; 104(1): Riemenschneider MJ, Jeuken JW, Wesseling P, Reifenberger G. Molecular diagnostics of gliomas: state of the art. Acta Neuropathol Nov; 120(5): Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, et al. 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