Low c-Met expression levels are prognostic for and predict the benefits of temozolomide chemotherapy in malignant gliomas

A total of 885 patients with 486 grade II, 202 grade III, and 197 grade IV primary gliomas were included in the immunohistochemistry study. All of these patients were managed with surgical resection and subsequent chemoradiotherapy and were independently re-evaluated by 2 experienced neuropathologists who were blinded to the clinical outcomes of the patients, according to the World Health Organization 2007 criteria. This study was approved by the Ethics Committee of Beijing Tiantan Hospital, the methods were carried out in accordance with the approved guidelines, and written informed consent was obtained from all patients included in this study. ¹⁷

Maximal tumor resection while preserving the key eloquent cortex was the principle goal during surgery. The extent of resection was assessed on the postoperative enhanced MRI within 72 h and graded as gross total or subtotal resection. Patients subsequently underwent concomitant TMZ and RT or radiotherapy in addition to concomitant and adjuvant TMZ chemotherapy (RT + TMZ) or postoperative radiotherapy only (RT only). Postoperative adjuvant radiotherapy was routinely delivered to the patient within four weeks after surgery. The total dose was 54–60 Gy, which was divided into 30 daily fractions of 1.8–2 Gy each, and five fractions were administered per week. For the patients who received adjuvant chemotherapy, the treatment was administered four weeks after radiation, and at least two cycles of chemotherapy were administered. Concomitant chemotherapy consisted of oral TMZ at a daily dose of 75 mg/mthat was given seven days per week from the first to the last day of radiotherapy for at most 49 days. After a four-week break, the patients received adjuvant oral TMZ (150–200 mg/m2) for five days every 28 days. A total of six cycles of TMZ chemotherapy were administered if no disease progression or irreversible hematological toxic effects were observed. ¹⁸

Survival data were collected in the clinics during the patient visits. The patients who underwent only tumor biopsy were not followed up at our center and were therefore excluded from the survival analysis. The baseline examinations included CT and magnetic resonance imaging (MRI), full blood counts and blood chemistry tests and physical examinations. During radiotherapy (with or without TMZ), the patients were seen every week. Twenty-one to 28 days after the completion of radiotherapy and every three months thereafter, the patients underwent comprehensive evaluations that included physical examinations and radiologic assessments of the tumors. During the adjuvant TMZ therapy, the patients underwent monthly clinical evaluations and comprehensive evaluations at the end of cycles 3 and 6. Overall survival (OS) was defined as the time between surgery and death. Progression-free survival (PFS) was defined based on the RANO criteria. ¹⁹

We used the whole genome mRNA expression microarray data for all tumor grades from Chinese Glioma Genome Atlas (CGGA) database() as a validation platform. The US National Cancer Institute Repository for Molecular Brain Neoplasia Data (REMBRANDT,),Data() and the Cancer Genome Atlas (TCGA) Database() were used to validate the mRNA and protein sets. ^{20 21 22} http://www.cgga.org.cn↗ http://caintegrator-info.nci.nih.gov/rembrandt↗ GSE16011 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16011↗ http://cancergenome.nih.gov↗

All of the materials were selected for DNA extraction via an elaborate examination of corresponding hematoxylin and eosin-stained sections. Every selected sample contained at least 80% of the vital tumor and was quick-frozen in liquid nitrogen and maintained at −80 °C. Genomic DNA was extracted from the frozen tumor tissues using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. The genomic region spanning wild-type R132 of IDH1 was analyzed with pyrophosphate sequencing using the following primers: (forward) 5′-GCTTGTGAGTGGATGGGTAAAAC-3′ and (reverse) 5′-biotin-TTGCCAACATGACTTACTTGATC-3′. For O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, bisulfite modification of the DNA was performed using an EpiTect Kit. The following primers were used to amplify the MGMT promoter region: (forward) 5′-GTTTYGGATATGTTGGGATA-3′ and (reverse) 5′-biotin-ACCCAAACACTCACCAAATC-3′.

Academy of Science. The cells were cultured in DMEM containing 10% FBS, 50 U/ml penicillin G and 250 μg/ml streptomycin in a humidified atmosphere containing 5% COat 37 °C. H4 and U87 cells were transfected with c-Met shRNA (3 target-specific plasmids, GeneChem, China, MET-RNA1 (15908-1), MET-RNA2 (16973-1), and MET-RNA3 (16985-1) and a control vector) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The MET-RNA1 target sequence was 5′-CTTATATGAAGTAATGCTA-3′, the MET-RNA2 target sequence was 5′-ACGATGAATACATTGAAAT-3′, and the MET-RNA3 target sequence was 5′-AACTTCTTTGTAGGCAATA-3′. The human glioma cell lines H4 and U87 were purchased from the Institute of Biochemistry and Cell Biology, Chinese 2

Whole cells were washed with cold PBS and lysed in RIPA buffer. Equal amounts of total protein (30 μg) from cell lysates were loaded onto 10% SDS/PAGE gels, transferred to PVDF membranes (Millipore), and detected using an ECL Western Blotting Detection System (Bio-Rad). The antibodies used were c-Met (1:2000 dilution, EP1454Y, Abcam) and GAPDH (1:5000 dilution, Proteintech).

For the MTT assays, the cells, including all transfectants, were grown to the exponential phase and detached by trypsin treatment. A total of 2000 cells/well were plated onto 96-well tissue culture plates (100 μl complete medium/well) and cultured at 37 °C in 5% CO. Different TMZ concentration gradients were performed on H4 and U87 cells, and after 3 days, 20 μL per well of MTT reagent was added and incubated at 37 °C for 1 hour. Subsequently, the absorbance values of each well were measured with a microplate spectrophotometer at 490 nm. Results were plotted as means ± SD of three independent experiments. 2

24-well chambers with 3.0 μm pore size (Corning) were used in cell migration and invasion assays. Cells were resuspended in 500 μL serum-free medium and 100 μL (about 100,000 cells) were seeded into the upper chamber (without or pre-coated with 200 ng/ml Matrigel solution (BD Biosciences, Bedford MA 01730, USA) in migration or invasion assay separately), 600 μL of DMEM containing 10% fetal bovine serum was placed in the lower chamber. After 24 h of incubation, the upper chambers were removed from the plates and cells on the top side of the chamber were wiped with a cotton swab. Migrating or invading cells were fixed and then stained. Four randomly fields were counted under a microscope and photos were taken.

Tumor samples collected during surgery or biopsy were embedded in paraffin and evaluated by experienced neuropathologists. One appropriate block from each specimen was selected, and serial 4 μm-thick sections of tissue were used for the immunohistochemical analyses of c-Met expression. In brief, immunoperoxidase staining for c-Met (Santa Cruz Biotechnology, sc-161) was performed following the standard protocol recommended by the manufacturer to show membrane and cytoplasmic staining. Each slide that was stained for c-Met was individually reviewed at 200x magnification and scored by two independent observers. Discrepancies in scoring between the two observers were resolved by an additional review of the specimens and a discussion was held between the reviewers until a consensus was achieved. The proportions of positively stained tumor cells were graded as follows: 0, no positive tumor cells; 1, <10% positive tumor cells; 2, 10–30% positive tumor cells; and 3, >30% positive tumor cells. The intensity of staining was recorded on the following scale: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). The staining index was calculated as follows: staining index=staining intensity×tumor cell staining grade. High c-Met expression was defined as a staining index score ≥4, and low expression was defined as a staining index <4.

Descriptive statistics, included counts, mean values, and standard errors, were calculated, as appropriate. The differences in c-Met expression were examined with ANOVA. The OS was calculated from the date of surgery to the date of the last follow-up or the date of death. The PFS was calculated from the date of the first surgery until the recurrence or the last follow-up. Survival curves were drawn using the Kaplan–Meier method, and the differences between the groups were compared with log-rank tests. P-values < 0.05 were considered statistically significant. Univariate analyses and multivariate analysis were performed with the Cox regression model. The factors that exhibited no significant association were eliminated (P > 0.05), and the remaining factors (P < 0.05) that were entered into the multivariate analysis were assumed to be independent predictors of survival. All data analyses were performed with GraphPad Prism, R and SPSS (Statistical Package for the Social Sciences) software.

For immunohistochemistry, we selected a patient population with available concrete clinical information that included 429 grade II, 179 grade III, and 175 pGBMs for our training set. The characteristics of patients with grade III and primary GBM are given in. Regarding the mRNA platform, among the data from the 286 glioma patients whose medical records were obtained from the CGGA database, there were 126 grade II gliomas, 51 grade III gliomas and 109 pGBMs. The other 264 and 317 samples from theand Rembrandt databases, respectively, were used as validation sets. Table 1 GSE16011

The expression of c-Met was measured in a series of 885 glioma samples by IHC (). As shown in, the pGBMs and anaplastic gliomas exhibited significantly higher levels of c-Met expression than the low-grade gliomas (P < 0.0001). To further confirm this result, the other 3 validation data sets were examined, and the microarray results revealed that the pGBMs exhibited a significant increase in c-Met transcript level compared with the mean expression levels observed in the low-grade gliomas and anaplastic gliomas (P < 0.0001;). Fig. 1E Fig. 1A Fig. 1B–D

To achieve stratification based on c-Met protein expression, the patients were divided into high c-Met expression and low c-Met expression groups based on the staining indices. The mean overall survival times of the pGBM and grade III patients with high c-Met expression were obviously shorter than those of the corresponding patients with low c-Met expression (P = 0.01 and P = 0.01, respectively, by log-rank test;). The mean progression-free survival time of pGBM and grade III patients with low c-Met was significantly longer than that of patients with high c-Met expression (P = 0.01 and P < 0.01, respectively, by log-rank test;). Subsequently, these results were confirmed based on the mRNA levels in the three validation sets (). To determine the prognostic value of c-Met in high-grade gliomas, we conducted univariate Cox regression analyses using the clinical and genetic variables of the high-grade patients. We found that age, preoperative KPS score, extent of resection, radiotherapy, chemotherapy, MGMT promoter methylation, and c-Met expression were statistically associated with the OS of the pGBM patients. Next, multivariable regression analysis was applied to evaluate the independent prognostic values of these clinicopathologic factors for patient survival. Ultimately, all of the data revealed that c-Met expression was an independent prognostic factor when age, preoperative KPS score, extent of resection, radiotherapy, chemotherapy, and MGMT promoter methylation were considered (). Unexpectedly, the same result was not observed in the grade III glioma patients (data not shown). Fig. 2 Fig. 2 Figure S1 Table 2

In our research, the patients who received aggressive therapy achieved the best OS and PFS, and the patients with pGBMs who received only radiotherapy exhibited the worst survival (,; All P < 0.0001). To further explain this phenomenon, we postulated an association between c-Met expression and therapeutic outcome and divided the patients into high-expression and low-expression groups and then further subdivided these groups into alkylating agent-treated and non-alkylating agent-treated subgroups. We found that the patients in the low-expression group were perfectly separated, which indicates that the patients who accepted aggressive therapy experienced longer OS (, P < 0.0001) and PFS (, P < 0.0001) than the patients who received only radiotherapy but that the same phenomenon was not observed in the high-expression group (, P = 0.2833;, P = 0.0687). The same method was also applied to the whole-genome mRNA expression microarray data and the same phenomenon was observed (). Fig. 3A D Fig. 3B Fig. 3E Fig. 3C Fig. 3F Figure S2

To underscore the potential for a medication-guiding role for c-Met, we first downregulated the expression of c-Met in two glioma cell lines that originally expressed c-Met at high levels. We followed this with a functional assay. Compared with control shRNA, MET-RNA2 clearly decreased the protein expression of c-Met (), and H4 and U87 cells presented inhibited proliferation with TMZ-treatment in the absence of c-Met () according to an MTT assay, whereas application of c-Met with control shRNA had a reduced effect. The same result was also verified by analysis of mRNA and protein levels (). Consequently, inhibiting MET enhanced the effect of TMZ sensitivity in both cell lines. Fig. 4A Fig. 4B Figure S3

The migration assay and Matrigel invasion assay were performed using U87 and H4 cells. The migration assay showed that remarkable cell migration diminution in c-Met-shRNA group compared with in the control shRNA group (). The Matrigel invasion assay also showed numbers of invading cells in the control shRNA group were significantly increased compared with the c-Met-shRNA group (). Fig. 5 Fig. 5