High prevalence of clonal hematopoiesis‐type genomic abnormalities in cell‐free DNA in invasive gliomas after treatment

Abstract Plasma cell‐free DNA (cfDNA) is emerging as an important diagnostic tool in cancer. However, cfDNA alterations may differ from those in tissue and sometimes may reflect processes unrelated to the cancer, including clonal hematopoiesis (CH). We examined plasma cfDNA, tested by next‐generation sequencing (NGS), for characterized alterations (excluding variants of unknown significance) in 135 patients with invasive glioma. Overall, 21% (28/135) had ≥1 alteration; 17% (23/135) had CH‐type cfDNA mutations. Temozolomide (a mutagenic alkylating agent) with concurrent radiation therapy prior to blood draw was significantly associated with an increase in CH‐type mutations, even after age, race/ethnicity, and WHO‐grade were considered as confounders (odds ratio [95% confidence interval, CI] 8.98 [1.13‐71.46]; P = .04; multivariable analysis). Further, of 18 patients with invasive glioma who had both cfDNA and tissue DNA NGS and had ≥1 cfDNA alteration, 16 (89%) had ≥1 cfDNA alteration not found in their tissue DNA, including CH‐type alterations in genes such as TP53 (most common), ATM, GNAS, and JAK2. Altogether, 87% of cfDNA alterations (20/23) observed in the 18 patients were implicated in CH. Finally, examining all 135 patients, CH‐type cfDNA mutations were an independent prognostic factor for shorter survival (hazard ratio [95% CI] 3.28 [1.28‐8.40]; P = .01). These findings emphasize that not all characterized cfDNA alterations detected in patients with solid tumors are cancer‐related. Importantly, in patients with invasive gliomas who have had prior temozolomide and radiation, CH‐related alterations in cfDNA are frequent and correlate with poor outcomes.


| INTRODUCTION
Despite advances in the therapeutics of diverse cancers, treatment options for progressive brain tumors remain limited. Glioblastoma multiforme (GBM), in particular, has a dismal prognosis, with a 10% survival at 5 years and a median survival <15 months, even after the introduction of temozolomide (TMZ). 1 There is an unmet need to better understand the biology of these lethal cancers. Repeated tumor tissue biopsy for sequencing is difficult in brain tumors and less-invasive molecular profiling using plasmaderived cell-free DNA (cfDNA) is now promising. In several tumor types, the prevalence of characterized cfDNA alterations is 60% to 80% and concordance rates between cfDNA and tissue characterized alterations have been reported to be from 50% to 90%. [2][3][4][5][6] In respect to brain gliomas, it has been suspected that leakage of DNA from intracranial lesions into the peripheral circulation may be somewhat attenuated by the blood-brain-barrier and that detectable cfDNA in patients with brain tumors is lower than that in other types of tumors. However, several studies have shown that a subset of patients harbor detectable cfDNA. [7][8][9] Even so, the data regarding liquid biopsy in patients with brain gliomas remain remarkably sparse.
There are many reasons that could account for differences in alterations between cfDNA and tissue: (a) tissue sequencing examines only the small piece of tissue sequenced while cfDNA may detect shed DNA that reflects tumor heterogeneity from multiple metastatic sites; (b) cfDNA may be suppressed by therapy; (c) cfDNA is present in small amounts and requires sensitive techniques; and (d) cfDNA (but also possibly tissue) may reflect detection of alterations due to clonal hematopoiesis (CH). 10 14,15 In general, the prevalence of CH mutations increases with age and is over 7% in people aged 60 years or older without a detectable solid or hematologic malignancy. [15][16][17] Also, CH mutations occur more frequently in white race. 15 In patients with cancer, certain types of environmental insults, such as exposure to chemotherapy or radiation, can increase the frequency of genomic mutations. 18,19 In fact, it has been reported that CH mutations were detected in 25% of patients with cancer (who did not have hematologic malignancies). 15 In our study, we analyzed cfDNA samples from 135 patients with invasive gliomas, defined as diffuse astrocytic or oligodendroglial tumors, and assessed the potential impact of CH-type mutations on cfDNA sequencing and correlation with clinical outcomes.

What's new
Tissue biopsy for brain tumors presents significant challenges, making less-invasive molecular profiling with plasma cell-free DNA (cfDNA) an appealing alternative. However, whether alterations detected in cfDNA, including clonal hematopoiesis (CH), reflect processes in brain tumor tissue remains uncertain.
In this investigation of CH-associated mutations in plasmaderived cfDNA from patients with invasive gliomas, 87 percent of characterized cfDNA alterations were implicated in CH, indicating that not all cfDNA alterations are cancer-related. In addition, temozolomide/radiation therapy prior to blood draw for cfDNA was associated with potential CH-type cfDNA mutation detection. Potential CH-type cfDNA alterations were an independent predictor of shorter overall survival. of mutant molecules divided by the total number of DNA fragments in each mutated gene.

| Tissue-DNA
For comparisons with cfDNA, tissue-DNA NGS was also used if available.       The bold values represent the statistically significant P-values (<.05). b At the time of blood draw for cfDNA (years), age 56 years is the median and was chosen as the cutoff; other cutoffs, for example, 65 or 70 years, were also not significant (P = .54 and P = .69, respectively).

| Definition and statistical analysis
were observed in seven patients (70%) (MET amplification and alterations in BRAF, BRCA1, FBXW7, and NF1 were not considered as CH) (

| The percent of patients with CH-type mutations increased after therapy with temozolomide and radiation
In this series, 101 of 135 patients (75%) received temozolomide with concurrent radiation therapy (TMZ + RT) prior to blood draw for cfDNA.
Patients who received TMZ alone (N = 5) or radiation alone (N = 5) prior to blood draw for cfDNA were treated as those "without TMZ + RT" in the analyses. Detection rate of characterized cfDNA alterations was sig- advance diverse cancers, the specificity (false positive) of cfDNA NGS reports is becoming an important issue to be discussed. "False positive" cfDNA alterations may reflect alterations not present in the tumor, but rather CH-type mutations, which are associated with blood cells derived from a hematopoietic stem cell and often detected even in people without any hematologic malignancies. [14][15][16][17] In general, characterized cfDNA alterations are often considered targetable by anticancer drugs in the precision therapeutic approaches for patients with solid tumors, but CH-type mutations should be removed from the therapeutic equation.
In this series, 21% of 135 glioma patients have at least one characterized alteration in peripheral blood-derived plasma cfDNA NGS ( Figure 1A). The rate was consistent with the 10% to 29% described in previous reports. 8,23 The subset of patients with detectable characterized cfDNA increased with WHO-grade: 23% to 24% in grade III and IV (AO, AA, or GBM) vs 6% in grade II (DA or OD) (P = .20), albeit not statistically significant ( Figure 1B). The characterized cfDNA alterations were seen in the 12 genes listed in Figure 2A, and the most common alterations in cfDNA were in TP53 (13%) followed by JAK2 In the patients who had both cfDNA and tissue DNA NGS performed and whose cfDNA showed ≥1 characterized alteration, characterized cfDNA alterations not found in tissue DNA could be seen in 94% of patients (Table S3). These cfDNA alterations were comprised of ATM, GNAS, JAK2, PDGFRA, and TP53 mutations. We considered that the mutations in ATM, GNAS, JAK2, and TP53 genes (but not the PDGFRA mutation in ID#50) were possibly CH-type mutations (Table S3). Interestingly, the common cfDNA alterations in patients with invasive glioma were mostly implicated in CH. In general, ASXL1, DNMT3A, PPM1D, and TET2 genes are commonly involved in CH, but these were not included in the sequencing cfDNA panels of the current study. Also, a previous study suggested that not all CH variants can be detected in the blood cell-derived DNA. 24 Although it is difficult to determine if these mutations are CH or cancer-related, over 80% of the invasive glioma patients with characterized cfDNA alterations detected had at least one potential CH-type mutations in our analysis.
We also observed that the prevalence of characterized cfDNA alterations in patients with invasive gliomas was significantly higher in blood samples biopsied after TMZ + RT (27% vs 2.9%, P = .003) ( Figure 1C).  15,25,26 TMZ, an alkylating pro-drug, methylates DNA and leads to genomic instability and eventually cancer cell death, but also causes inactivating mutation in the mismatch repair genes, inducing hypermutations. 27,28 Similarly, radiation mutagenesis in which free radicals affect the cytoplasm and lead to DNA damage is well known. 29 In this series, MET amplification and mutations in PDGFRA, EGFR, BRAF, BRCA1, FBXW7, and NF1 were considered as non-CH type alterations.
Although cfDNA IDH1 mutations generally can be considered as potential CH-type, an IDH1 mutation in cfDNA of patient ID#105 was considered as a non-CH type alteration because her tissue DNA also showed the identical IDH1 alteration (Table S3)

DATA AVAILABILITY STATEMENT
The data that support the findings of our study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
This study was performed in accordance with the guidelines of the Internal Review Board-approved/declaration of Helsinki compliant study-UCSD-PREDICT (NCT02478931) and any investigational therapies for which the patient gave consent.