The molecular landscape of ctDNA in resectable gliomas.
Firstly, Blood samples from the 69 patients were sequenced to exclude somatic mutations. And the distribution of pathological types is shown in the figure1A, identifying radiological relapse in 44 patients (mean age, 52 years). In all 57 tumor sample, 199 mutations were identified: 12 patients (21%) with 1 mutation and 45 patients (79%) with multiple mutations using the 68-cancer gene panel. The most frequently detected mutation was TP53 (44%) followed by IDH1(39%) and PTEN (25%; figure1B), with median average VAF of 40.5%. We also compared the VAF of variants between tumor and TISF before postoperative treatment, in this cohort, we see a difference in VAF between these two sample types (P <0.0001), This could be due to a substantial reduction in tumor burden after surgery.
TISF from 29 patients (comprising 45 samples) at or after a diagnosis of recurrence was available and at least one variant was detected in 93% (27/29) of cases. There are 613 gene mutations were found, TP53 was also the most frequently mutated gene at this clinical timepoint (48%) ,next NF1 (38%) and PTEN (31%), mean VAF from 0.2%-34.14%, with a median 1.54%(figure1C). Of the two cases in which a variant was not detected at or after recurrence, no variants were detected at any point in the cases during their clinical pathway, indicating these patients had low shedding tumors. One of the patients did not detect any mutations in the TISF obtained the day before the second surgery, and the pathological diagnosis after the operation was confirmed to be gliosis, and we considered it to be a Pseudoprogression.
Baseline TISF ctDNA and Prognosis
23 patients received TISF for ctDNA sequencing 9-43 days（median follow-up of 23 days）after surgery, excluding somatic mutations from the blood and 23 TISF(+),MRD was detected in 23/23 patients at a median allele frequency of 0.45% . In 31 tumor samples obtained at operation, mean allele frequency from 2.1%-79.15%，median allele frequency of 39.52%，mean allele frequency was associated with relapse-free survival (hazard ratio, 2.214; 95% CI, 1.003 to 4.887; P = 0.0165)(figure 2A、B)
To evaluate the role of TISF ctDNA in recurrence surveillance after definitive operation, we obtain TISF samples for ctDNA sequencing 100-300 days after operation, which gave rise to a ctDNA analysis subgroup of 14 patients. We found that the mean VAF is 0.21%-20.825%，median allele frequency of 39.52%.Remarkably, higher than median VAF has a very high risk of recurrence(hazard ratio, 4.058; 95% CI, 1.089 to 15.12; P =0.0012) (figure 2C、D).
ctDNA fraction and response to TMZ treatment
There was statistically significant difference in tumor and baseline ctDNA fraction (p<0.0001). The ctDNA fraction among nonresponding patients (determined via serial radiological) was relatively unstable throughout treatment (mean 1.01% at baseline, 11.735% on treatment, and 3.12% at EOS) (Fig 3A,B). By contrast, the ctDNA fraction among responders exhibited stable on treatment(mean 0.399% at baseline, 0.562% on treatment, and 2.848% at EOS). We further evaluated the ctDNA fraction in longitudinal same-patient samples (Fig 3C,D). Among responders, no significant change on treatment and increased at EOS (mean 0.2% at baseline, 0.47% on treatment, and 5.7% at EOS); this was not observed for nonresponders(mean 0.29% at baseline, 15.94% on treatment, and 0.99% at EOS)
Early detection of hypermutation in TISF ctDNA and in vivo evolution of hypermutation
We next explored early detection of hypermutation in TISF ctDNA and in vivo evolution of hypermutation. A total of 3/69(4.3) hypermutated patients with 132-189 mutations were identified in our cohort, and we tracked the in vivo evolution of hypermutation in 2/3 by serial TISF ctDNA monitoring.
In all 3 TISF samples at the time of hypermutation, we found that in addition to the large number of mutations, there was MMR gene deficiency with a large number of point mutations characterized by G:C>A:T, consistent with signature 11, and in tumor tissue no MMR-related gene mutations were found in all of them, which were confirmed to be tumor hypermutations induced during TMZ treatment.
In one of the patients with continuous TISF surveillance, we found PMS2: p.A127T 232 days before hypermutation, but the mutation frequency was only 0.1%. In TISF samples after 112 days, we found mutations at different sites such as MSH2, MSH6, MLH1, etc. However, the number of mutations at this time was 22, indicating that hypermutation had not occurred (Tumor VS TISF1 paired t-test showed statistical difference, P <0.0001; Tumor VS TISF2, P =0.0451; TISF1 VS TISF2, P =0.0099). In addition, we detected in TISF samples After 63 days of hypermutation, radiographic recurrence was found. In the other patient, we obtained only one TISF sample before hypermutation, with an interval of 303 days, so no MMR-associated mutation was detected (Tumor VS TISF1, Tumor VS TISF2, Paired t-test showed no statistical difference; TISF1 VS TISF2,P=0.0020).