2.1 Patient characteristics

We prospectively collected tumor specimens, matched TISF samples, and blood samples from 11 patients with oligodendroglioma, World Health Organization (WHO) grade II-III, with IDH mutation and 1p19q co-deletion. All patients were treated at Henan Provincial People's Hospital in China from July 2014 to January 2022. This work was supported by the Zhengzhou University, and all patients provided written informed consent to participate in the study and for use of their clinical data and specimens for research. This study was performed according to the research proposals approved by the Institutional Review Board of Henan Provincial People's Hospital and the Ethics Committee of the Zhengzhou University (approval number 2122432055). We reclassified all tumors according to the 2021 update to the WHO classification of tumors of the central nervous system.² Histopathologic classification and molecular information were reviewed and confirmed to correspond to oligodendroglioma. Relevant clinical information, including age, gender, tumor location, postoperative therapy, and others, was obtained. Table S1 shows a summary of the respective patient dates.

2.2 Sample collection

TISF samples were harvested as previously described.¹² We obtained a small amount of TISF from the implanted reservoir (intraoperative retention). TISF is fluid in the local surgical cavity, which may support in situ detection of gene evolution during oligodendroglioma progression. At the same time, blood samples (5 mL) for germline DNA control were also acquired. ctDNA profiling from tumor tissues and TISF samples has utility for real-time assessment of dynamic tumor evolution.

2.3 DNA extraction

Cell-free DNA (cfDNA) was extracted from TISF samples using the Cell-Free DNA Isolation Kit (Thermo Fisher Scientific). While glioma genomic DNA was extracted from fresh tumor tissue using the QIAamp DNA Tissue & Blood Kit (Qiagen). Control genomic DNA was prepared from paired blood samples using the QIAamp DNA Mini Kit (Qiagen). All DNA extractions were performed with the respective kit according to the manufacturer's protocol. The DNA concentration of each sample was measured using a Qubit assay kit (Thermo Fisher Scientific).

2.4 ctDNA sequencing

DNA sequencing was performed and analyzed by Beijing Genetron Health Technology Co., Ltd. Briefly, genomic DNA was PCR amplified using a 10 ng of genomic DNA primer pool as a template. Oligodendroglioma samples underwent high-throughput exome sequencing with a targeted deep sequencing assay of 68 genes recurrently mutated in brain tumors (Genetron Health). Sequencing data were processed using the Torrent SuiteTM software 4.4 and quantified using the Torrent Mapping and Alignment Program (TMAP) algorithm (Life Technologies).The variants were detected using the Variant Caller Plugin v4.4 (Life Technologies). We considered a mutant allele fraction of ≥0.3% and a total of ≥4 reads detectable in samples. Finally, the alignment files were normalized and checked using deeptools and visualized with the Integrative Genomics Viewer (IGV) software. Resulting sequence reads were aligned against the reference DNA sequence, and variants were analyzed by using the Next GENe® v.2.3.4 software (SoftGenetics). All detected mutations were annotated for genes using ANNOVAR, Oncotator, and Vep.

2.5 1p/19q status

Determination of 1p/19q co-deletion status was performed locally by dual-color fluorescence in situ hybridization (FISH) analysis.¹⁴ Briefly, paraffin-embedded tumor samples were sectioned and deparaffinized. The labeled probes mixed with Cot-1 DNA (Life Technologies) were denatured and hybridized to sections. After washing with PBS, the sections were incubated in anti-digoxigenin antibody and fluorescein isothiocyanate-conjugated secondary antibody. Sections were mounted with mounting medium and viewed under a Zeiss microscope (CarlZeiss Microscopy LLC).

2.6 Statistical analysis

We compared the VAFs of mutations between the primary cancer and disease progression. Statistical analysis was performed using GraphPad Prism 8.0. Comparison between two samples was performed using an independent sample t-test. p < 0.05 was considered statistically significant.

3.1 Characteristics of oligodendroglioma cases

To elucidate the mechanisms driving the evolution of oligodendroglioma under TMZ therapy, we analyzed exome sequencing data from 11 oligodendrogliomas. These cases harbored IDH1 mutations and 1p/19q co-deletion: three patients with WHO grade II and eight patients with Grade III histology. After surgery, all patients were treated with TMZ treatment and five patients received radiotherapy treatments. Of the six patients who had a recurrence, one patient had local recurrence only and five patients had distant recurrence. Patients 24 and 27 received long-term TMZ treatment before the surgery (Figure 1A).

We performed exome sequencing of 11 tumor samples and 11 matched normal blood samples. The sequencing data that include oligodendroglioma driver genes are summarized in Figure 1B. Among them, nine cases had no treatment before operation. Overall, our mutational analysis reveals both known and potentially driver gene mutations in oligodendroglioma. We observed major driver gene mutations known in oligodendroglioma, including CIC (6/9), FUBP1 (3/9), TP53 (3/9), Notch1 (3/9), KRAS (2/9), PIK3R1 (1/9), BOCR (1/9), SMARCB1 (1/9), and ATRX (1/9), accompanying with IDH-mutant and 1p/19q co-deletion (Figure 1B). We have no information for TERT promoter mutations due to exome sequencing. But long-term exposure to therapeutic concentrations of TMZ before surgery, more mutations in SHh (Sonic hedgehog) and PI3K/AKT signaling pathways appeared, including PIK3CA (1/2), AKT1 (2/2), PTCH1 (2/2), and PTPN11 mutations (2/2). Even Patient 24 had a YAP1 mutation associated with the Hippo signaling pathway (Figure 1B). We show that a complex branching evolutionary pattern occurs in oligodendroglioma, and that clonal evolution can be influenced by TMZ treatment.

In addition, we further investigated mutation sites in primary tissues, which makes observation of timely tumor progression possible. R132H mutation of IDH1 is the most frequent genetic alteration in oligodendroglioma. IDH1:p.R132H mutation results in a gain of enzyme function and is thought to promote gliomagenesis.¹⁵ CIC:p.R215W mutation is a common mutation in oligodendroglioma, which leads to the loss of CIC protein function,¹⁶ while we observed CIC:p.K1517del was the most frequent mutation in our group (Figure 1C). In addition, under long-term TMZ treatment, Patients 24 and 27 acquired different mutation sites in the same genes, such as TP53, NOTCH1, CIC, and FUBP1, in which mutations were observed in primary tumors. It is usually observed that the two patients acquired more mutations, and emerging evidence suggested that alterations in genes vary substantially, indicating spatiotemporal heterogeneity.

3.2 Recurrence mutation analysis

We performed exome sequencing on tumor samples and matched real-time TISF samples in vivo from six patients with recurrence. A total of 22 tumor and TISF samples were detected from these patients. The mutation list and detailed information can be found in the attached file Figure S1. Four patients had not received any treatment prior to surgery, and the average number of non-synonymous mutations was 3.5 (ranging from 2 to 4). Two cases had received long-term TMZ chemotherapy. The average number of non-synonymous mutations in tumors was 19.5 (18, 21) (Figure 2A). And the retention rate, which is the ratio of primary tumor mutations retained in relapse tumors,¹⁷ was generally low (Figure 2B), with an average of 26.8% (ranging from 0 to 100), indicating that spatiotemporal heterogeneity may contribute to disease progression. Meanwhile, the gene evolution presented a large difference among different patients. Interestingly, there was one case in which the recurrent TISF sample shared the same mutations with the primary tumor (Figure 2A, Patient 28). Different mutations of the same gene, including CIC mutation in patient 63 and Notch1 mutation in Patient 24, were observed in recurrent TISF, which proved the convergent evolutionary model (Figure 2A). In order to infer the advantage of each mutation for tumor growth, we focused on the retained or acquired mutations in recurrent samples. In some cases, even the well-known putative major driver gene mutations in CIC and FUBP1 detected in primary tumors were not detected in recurrence TISF samples (Patients 29 and 30), and the FUBP1 mutation was obtained with a low VAF of 0.3% in one case (Patient 63). It can be seen that CIC and FUBP1 mutations may have limited effects on promoting the progression and recurrence of oligodendroglioma. Furthermore, the IDH mutation status also changed, as it was not detected in recurrence samples (Patients 29 and 30) (Figure 2A). IDHmt-to-IDHwt transformation indicated that the cell phenotype and genetic characteristics of oligodendroglioma changed during tumor evolution, which promoted tumor progression into its more aggressive form.

Two patients received long-term TMZ chemotherapy before surgery, and mutations in the SHh (PTCH1) and PI3K/AKT signaling pathways (AKT1, PIK3CA, PTPN11) were detected in two tumor tissues (Patients 24 and 27) (Figure 2C,D). Mutations in MMR genes were obtained (Patient 24 had MSH2 and MSH6 mutations, and Patient 27 had an MSH6 mutation), and hypermutation occurred in Patient 24 during gene evolution. Patient 27 acquired a Notch1 mutation at the time of tumor recurrence. We speculate that the Notch1 mutation is not only a driver gene, but also may contribute to tumor progression and recurrence. TP53 mutation was persistent in both the primary tumor sample and the matched-relapse TISF sample. In summary, our results indicated that oligodendroglioma has great heterogeneity during gene evolution and suggested that several well-known signaling pathways and genes play an important role in tumor evolution, including the SHh signaling pathway, PI3K/AKT signaling pathway, Notch1 signaling pathway, and MMR proteins (MSH2 and MSH6).

3.3 Gene evolution in distant recurrence oligodendroglioma

Our study indicated a higher proportion of distant relapse during oligodendroglioma evolution. To elaborate on the evolution pattern of distant relapse, we investigated continuous tumor genomic evolution through a series of TISF-ctDNA in vivo. Patient 29 was initially diagnosed with a lesion in the right temporal lobe by imaging, then underwent the surgery to resect the lesions, and was histopathologically diagnosed with anaplastic oligodendroglioma (WHO grade III). TP53:p.L194R, CIC:p.R1214*, and FUBP1:p.R344* mutations were detected as early driver genes, accompanied by IDH1:p.R132H mutant and 1p/19q-co-deleted. We collected TISF1 samples at 443 days after surgery, and none of the mutations with a VAF of ≥0.3% were detected. The result was consistent with the little progression of the tumor on imaging. The patient showed progressive symptoms 798 days after surgery, IDH1:p.R132H, TP53:p. R248W, PTCH1:p.G17del, and MYCN:p.P44L appeared in the TISF2 sample due to local progression. The patient continued to receive monthly TMZ, BEV, and MTX treatment. We collected TISF3 samples at 896 days after surgery and detected only one mutation (TP53:p.R248W) with a low VAF of 0.5%. The patient experienced distant recurrence in the corpus callosum approximately 3 years after surgery. NOTCH1:p.F937L with a VAF of 10.7% and TP53:p.R248W with a VAF of 0.7% were detected in the TISF4 sample (Figure 3A,B). As the disease progressed, the VAF value increased significantly (Figure 3C). It is obvious that oligodendroglioma displayed branched clonal evolution leading to tumor heterogeneity. Our study demonstrated that the emergence of subclones and IDH mutation status transformation promoted tumor malignancies and recurrence.

Patient 27 with a lesion in the right frontal lobe underwent surgery in 2014 and had a pathological diagnosis of oligodendroglioma (WHO grade II). The patient received long-term TMZ treatment and underwent a second resection for a known local recurrence in 2018. The pathological diagnosis was anaplastic oligodendroglioma (WHO grade III). Gene analysis showed CIC:p.F207I, TP53: p.G187D, and IDH1:p.R132H as driver genes, while subclonal mutations in SHh (PTCH1:p.P916L), Wnt (PTEN:N/A), and PI3K/AKT signaling pathways were clearly detected in tissue, and MMR-related subclone mutation was also found. PMS2 is a component of the post-replicative DNA mismatch repair (MMR) system. MMR dysfunction ultimately led to TMZ resistance,¹⁸ and lead to a large number of C > T/G > A mutations (Figure 3F). Two years later, the distant recurrence progressed, PIK3CA:p.E545K was detected in the TISF sample. PIK3CA:p.E545K was one of the most common activation mutations and promoted tumor formation¹⁹ (Figure 3D,E). Oligodendroglioma displayed remarkable spatiotemporal heterogeneity during tumor evolution, and MMR dysfunction largely contributes to the accumulation of mutations during tumor progression.

Patient 30 had a lesion in the left frontal lobe and underwent the first surgery to resect the lesion in 2018. The pathological diagnosis was anaplastic oligodendroglioma (WHO grade III), with IDH1:p.R132H, PIK3R1:p.G376R, CIC:p.N205S, and FUBP1:p.Y505Gfs*16 mutations confirmed as driver genes in tumor tissue. We collected TISF1 samples 16 months after surgery and confirmed one mutation (IDH1:p.R132H) with a VAF of 3.7% associated with local tumor progression. The patient received TMZ combined with MTX treatment. Twenty-six months after the first surgery, the patient underwent a second operation due to hematoma formation. However, our sequence analysis demonstrated that no gene mutations in the tissue were detected. We considered that the patient suffered from hemorrhage and did not appear to be due to tumor progression, and was likely to respond to chemotherapy. However, 39 months after the first surgery, the patient developed distant recurrence. Some subclonal mutations in PTCH1:p.R1350W and MYC:p.S207L appeared (Figure 3G,H). The MSAF of samples in the TISF samples increased as the tumor progressed (Figure 3I). Patient 63 acquired subclonal mutations in FUBP1:p.I271Rfs*3 and BCOR:p.D1355N when tumor recurrence was observed (Figure 3J). It can be seen that oligodendroglioma is sensitive to chemotherapy, but after long-term TMZ treatment, oligodendroglioma displayed obvious spatiotemporal heterogeneity, that is, acquired a large number of subclonal mutations during gene evolution, which promotes tumor distant recurrence.

3.4 Distant relapse may be a later event in oligodendroglioma

Among the six recurrent oligodendrogliomas, one developed local recurrence. Patient 28 underwent surgical resection and was diagnosed with anaplasia oligodendroglioma (WHO grade III). IDH1:p.R132H mutation and 1p/19q co-deletion were confirmed as driver events in the tumor tissue. Meanwhile, this patient had dominant mutations in TP53:p.R175H, TP53:p.R273C, and ATRX:p.W1572*. One year after the operation, imaging demonstrated local progression, and TISF1 sample was harvested at the time. We observed that the same mutations were present in both the initial and recurrent samples (Figure 4). The TP53:p.R175H mutation is a functional acquired mutation closely related to the high invasiveness and metastatic potential of many cancers.²⁰ TP53:p.R273C is also a functional acquired mutation that promotes the proliferation, invasion, and drug resistance of tumor cells.²¹ The 1p/19q co-deletion is positively associated with IDH mutations, while it is mutually exclusive with ATRX loss and TP53 mutation, which are the hallmarks of diffuse astrocytoma.²² Patient 28 had the molecular characteristics of oligodendroglioma and the molecular information of astrocytoma, and there was no tendency for an increase in mutation numbers. Subsequently, the local lesion improved significantly under TMZ treatment. Similarly, Patients 29 and 30 did not experience changes in IDH mutation status when local progression occurred (Figure 3B,H). However, under long-term TMZ chemotherapy, IDHmt-to-IDHwt transformation and additional subclonal mutations were present when distant relapse occurred and had a tendency to rapid tumor progression, indicating that the cell phenotype and genetic characteristics of oligodendroglioma will change under TMZ treatment. Oligodendroglioma not only has the characteristics of oligodendrocyte, but also has the molecular characteristics of astrocytoma in the samples from patient 28. The IDH mutation transformation suggests that relapse may emerge in the later stages of tumor evolution after diverging from the initial tumor ancestors. Distant relapse may be a later event during oligodendroglioma evolution.

3.5 Hypermutation in oligodendroglioma

Hypermutated tumors have been reported in patients with glioblastomas and astrocytomas,²³ but hypermutation has not previously been reported in the oligodendroglioma patients, even after 12 courses of TMZ chemotherapy.⁹ However, Patient 24 who had MMR-associated mutations, exhibited a hypermutable phenotype, which may have been related to TMZ treatment. Patient 24 was initially diagnosed with a lesion in 2015 and received long-term TMZ treatment. Unfortunately, multiple lesions were detected by imaging in 2020, and the patient underwent surgery, during which the tumor was diagnosed as oligodendroglioma (WHO grade III). We confirmed IDH, Notch1, CIC, and FUBP1 as the driver genes, accompanied by 1p/19q co-deletion (Figure 3E). Multiple events in signaling pathways could be seen (e.g., SHh, PI3K/AKT, and YAP1/Hippo signaling pathways), which contributed to the biological characteristics of distant relapse. The tumor remained stable, and accordingly, all mutations were detected with a VAF less than 1.5% in TISF1 samples. However, imaging revealed an increase in tumor burden at 138 days post-operation. Additionally, an MMR mutation in MSH2:p.Q337* was detected in the TISF2 sample, and the average VAFs in the TISF2 sample were significantly higher than those in the TISF1 sample. The patient then received TMZ and BEV treatment starting on day 157. The tumor load was significantly reduced, and the mutational burden decreased in the TISF3 sample. However, progression was noted again at 269 days after surgery, at which time, hypermutation occurred and 135 mutations were detected in the TISF4 sample (Figure 5B). And MSH6:p.H367Y and MSH2:p.A420T were included in the gene panel, and hypermutated tumor was more likely to progress rapidly and widely. The mean VAF in TISF4 was significantly increased again (Figure 5C). In addition, the proportion of total TMZ-associated mutations in the TISF4 sample was 94% (Figure 5D). There is evidence to suggest that the hypermutation is a contributor to malignant progression that can occur during the process of human oligodendroglioma evolution.