Maximum 11C-methionine PET uptake as a prognostic imaging biomarker for newly diagnosed and untreated astrocytic glioma

This study aimed whether the uptake of amino tracer positron emission tomography (PET) can be used as an additional imaging biomarker to estimate the prognosis of glioma. Participants comprised 56 adult patients with newly diagnosed and untreated World Health Organization (WHO) grade II–IV astrocytic glioma who underwent surgical excision and were evaluated by 11C-methionine PET prior to the surgical excision at Osaka City University Hospital from July 2011 to March 2018. Clinical and imaging studies were retrospectively reviewed based on medical records at our institution. Preoperative Karnofsky Performance Status (KPS) only influenced progression-free survival (hazard ratio [HR] 0.20; 95% confidence interval [CI] 0.10–0.41, p < 0.0001), whereas histology (anaplastic astrocytoma: HR 5.30, 95% CI 1.23–22.8, p = 0.025; glioblastoma: HR 11.52, 95% CI 2.27–58.47, p = 0.0032), preoperative KPS ≥ 80 (HR 0.23, 95% CI 0.09–0.62, p = 0.004), maximum lesion-to-contralateral normal brain tissue (LN max) ≥ 4.03 (HR 0.24, 95% CI 0.08–0.71, p = 0.01), and isocitrate dehydrogenase (IDH) status (HR 14.06, 95% CI 1.81–109.2, p = 0.011) were factors influencing overall survival (OS) in multivariate Cox regression. OS was shorter in patients with LN max ≥ 4.03 (29.3 months) than in patients with LN max < 4.03 (not reached; p = 0.03). OS differed significantly between patients with IDH mutant/LN max < 4.03 and patients with IDH mutant/LN max ≥ 4.03. LN max using 11C-methionine PET may be used in prognostic markers for newly identified and untreated WHO grade II–IV astrocytic glioma.

Although magnetic resonance imaging (MRI) has been one of the basic and less-invasive imaging modalities used in the management of glioma, brain PET imaging has recently been recommended 2,3 . We have previously reported a positive correlation between WHO grade and accumulation of 11C-methionine among astrocytomas, but that study did not analyze the relationship with prognosis 4 . Additional analysis was thus performed in the current study. Moreover, the clinical studies investigating the relationship between molecular analysis and uptake of amino acid PET in glioma patients are sparse, and detailed prognostic analyses of associations with molecular profiles and 11C-methionine PET uptake in glioma patients have not been fully completed. This study aimed to evaluate the association between 11C-methionine uptakes, and prognosis in cases of newly diagnosed and untreated adult astrocytic glioma.

Methods
Patients. From July 2011 to March 2018, there were 66 adult patients and two patients under 18 years old with newly diagnosed and untreated WHO grade II-IV glioma who underwent surgical tumor resection and preoperative 11C-methionine PET examination, as previously reported 4 . From this previous cohort, we included adult astrocytic glioma patients with IDH mutated-TERT promoter wild-type, or those with IDH wild-type in the present study. Finally, a total of 56 patients with astrocytic tumor were included in the present cohort. The 56 patients were comprised of 36 male and 20 female patients, with a mean age of 54.0 years (range, 21-82 years). All 11C-methionine PET was performed within one month prior to tumor resection in glioblastoma patients and within six months in patients with lower-grade glioma. Pathological diagnosis was determined according to the 2016 WHO classification for central nervous system tumors 5 . This study was approved by the institutional review boards at the Graduate School of Medicine, Osaka City University (Approval Numbers: 2047 and 2020-115), and Osaka National Hospital (Approval Number: 0713). Genetic analyses were performed after obtaining written consent. This study was complied with all tenets of the Declaration of Helsinki.
11C-methionine PET. An Eminence B PET scanner (Shimadzu, Kyoto, Japan) or Biograph-16 PET scanner (Siemens, Bon, Germany) was used for 11C-methionine PET, according to previously reported procedures 4, 6 . Mean and maximum lesion-to-contralateral normal brain tissue (L/N) ratios were determined by dividing the tumor standardized uptake value by the mean standardized uptake value of the normal contralateral region of the brain, as previously reported 4 .
Genetic analysis. Genetic analysis was performed as previously described 4 . Genomic DNA was extracted from surgically resected tumor specimens using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA) or NucleoSpin Tissue (Machery-Nagel, Duren, Germany). Hotspot mutations of IDH1/2 (codon 132 of IDH1 and codon 172 of IDH2) and TERT promoter (termed C228 and C250) were examined using Sanger sequencing with a 3130xLGenetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) and Big-Dye® Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA). The methylation status of O 6 -methylguanine-DNA methyltransferase (MGMT) promoter was analyzed using quantitative methylationspecific PCR after bisulfite modification of tumor genomic DNA, as previously reported 7 .
Survival times. Progression-free survival (PFS) was defined as the time in months between evaluation with 11C-methionine PET and tumor progression according to the Response Assessment in Neuro-oncology working group 8 . Overall survival was defined as the time in months between evaluation with 11C-methionine PET and death. Statistical analysis. Patients were subdivided into several groups on the basis of age (≥ 70 or < 70 years), preoperative KPS(≥ 80 or < 80), LN mean(≥ 2.46 or < 2.46), LN max(≥ 4.03 or < 4.03), and extent of resection (biopsy or partial removal, < 90%; subtotal removal, ≥ 90% or gross total removal, ≥ 95%) for statistical analysis.
To compare the patients background characteristics of each group classified according to IDH status or LN max or both, we performed statistical analysis using Pearson's chi-square test. PFS and OS were analyzed using the Kaplan-Meier method. Survival date were evaluated using univariate and multivariate Cox regression analyses. Prognostic factors with a p < 0.05 in the univariate analysis were included in the multivariate analysis. The stepwise method was used to evaluate PFS and OS multivariate Cox regression analyses. Statistical significance was defined at the level of p < 0.05. All statistical analyses were conducted using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan) 9 .
Ethical approval. This

Discussion
The revised WHO 2016 classification of the central nervous system tumor requires the pathological diagnosis with molecular analysis to reach a diagnosis of glioma 5 . This molecular information has been said to correlate with prognosis, whereas there is still a matter of debate whether imaging biomarkers help estimation of prognosis. Although MRI remains the gold standard for diagnosing glioma, its role in estimating prognosis is limited 10 . On the other hand, 11C-methionine PET using amino tracer might be useful to detect the tumor, predict the grade or genetic status or both 4,7,[11][12][13] , and distinguish tumor recurrence from radiation necrosis [14][15][16] in glioma patients, although 11C-methionine PET can only be used in limited institutions that have a cyclotron since 11C-methionine has a short half-life about 20 min. However, relatively few reports have investigated the relationship between the uptake of amino tracer using PET and prognosis in glioma. Moreover, reports investigating prognosis of glioma patients in association with molecular analysis and PET in glioma have been limited [17][18][19][20][21] . Thus, our goal in the present study was to determine whether 11C-methionine PET can be used as an additional imaging biomarker of prognosis.
In the present study, we excluded patients with oligodendroglioma, or those with IDH mutated-TERT promoter mutated, or both because oligodendroglioma is considered to show better prognosis than astrocytoma and is often accompanied by both IDH and TERT promoter mutations. Although TERT promoter mutation is often seen in oligodendroglioma and primary glioblastoma, prognoses differ markedly between oligodendroglioma and glioblastoma 22,23 . An argument has also been made regarding the association between uptake of 11C-methionine and oligodendroglioma 13,[24][25][26][27] . We have previously reported a positive correlation between WHO grade and the accumulation of 11C-methionine among astrocytomas, and a statistically higher uptake of 11C-methionine in oligodendroglioma than in diffuse astrocytoma 4 . In the current study, median PFS was 37.2 months for patients www.nature.com/scientificreports/ with diffuse astrocytoma, 9.6 months for those with anaplastic astrocytoma, and 4.7 months for those with glioblastoma, respectively. Median OS was not reached for patients with diffuse astrocytoma, 27.1 months for those with anaplastic astrocytoma, and 20.5 months for those with glioblastoma, respectively. Reuss et al. reported that 139 of 152 patients with diffuse astrocytoma diagnosed according to the WHO 2007 classification of the central nervous system tumors showed IDH mutant diffuse astrocytoma, whereas more than half of patients with diffuse astrocytoma were IDH wild-type in our cohort 28 . Minniti et al. reported that IDH mutant anaplastic astrocytoma was found in 56% of their anaplastic astrocytoma patients 29 . OS in patients with IDH wild-type was 2.8 years 29 . The relatively shorter PFS and OS of patients with diffuse astrocytoma and anaplastic astrocytoma in the current study were probably attributable to the fact that the present cohort included more patients with Table 3. Prognostic factors for PFS, and OS in the multivariate analyses KPS Karnofsky performance status, LN lesion-to-contralateral normal brain tissue, DA diffuse astrocytoma, AA anaplastic astrocytoma, GBM glioblastoma, IDH isocitrate dehydrogenase, TERT telomerase reverse transcriptase, MGMT O 6methylguanine-DNA-methyltransferase, Met methylation, CRT chemoradiotherapy, RT radiation therapy, Chemo chemotherapy, PR partial resection, STR subtotal resection, GTR gross total resection, PFS progressionfree survival, HR hazard ratio, CI confidence interval, OS overall survival. P values in bold font are statistically significant. Brain PET imaging has recently been recommended for use in addition to MRI in the management of glioma 2, 3 . Takano et al. reported that PFS was worse with LN max ≥ 2.0 than with LN max < 2.0 using 11C-methionine PET among patients with untreated, lower-grade, non-enhancing gliomas 31 . Discrimination of high-grade glioma from low-grade glioma is usually difficult using MRI alone prior to tumor resection in patients with non-enhancing, lower-grade glioma, so we considered whether 11C-methionine PET can be used to predict the prognosis of glioma. However, we could not find significant differences in PFS between astrocytoma patients with LN max ≥ 4.03 and LN max < 4.03 or between those with LN mean ≥ 2.46 and LN mean < 2.46 in the current study.
Recently, some reports have investigated the relationship between prognosis from molecular analysis and uptake of PET using 18F-fluoro-ethyl-tyrosine (18F-FET) PET 17-19, 32 and 3,4-dihydroxy-6-18F-fluoro-ethyl-Lphenylalanine (18F-FDOPA) PET 33 . Galldiks et al. in a study of photopenic IDH mutant gliomas reported that glioma with 18F-FET accumulation below the level of background healthy brain showed unfavorable outcomes, and thus should be treated more actively 18 . The utility of dynamic 18F-FET PET has also been reported 19 . Suchorska et al. reported that longer minimal time-to-peak analysis using 18F-FET PET was associated with a favorable prognosis in IDH mutant astrocytomas 19 . A time-to-peak analysis≥ 25 min was associated with longer PFS and OS in patients with IDH wild-type high-grade astrocytoma according to Bauer et al. 32    Some limitations need to be considered for the current study. First, the relatively small cohort of the current study might have influenced statistical analyses. For example, TERT promoter status did not influence OS in our cohort, although Arita et al. reported the usefulness of TERT promoter status in addition to the IDH status 34 . Further study with a larger cohort is thus needed to assess the correlation between prognosis and molecular/ imaging biomarkers with amino-tracer PET in patients with astrocytoma. Second, we did not take volumetric analyses into consideration in the current study, although some reports have suggested that metabolic tumor volume did not correlate with survival outcomes 17,19,32,33,35 .

Conclusion
LN max using 11C-methionine PET offers a markers for estimating OS in patients with grade II-IV astrocytoma. LN max can also be used as a prognostic imaging biomarker to estimate OS in addition to IDH status in IDHmutated astrocytoma.

Data availability
The date in the current study are available from the corresponding author on reasonable request.