CfDNA-based liquid biopsy of cerebrospinal fluid in medulloblastoma

FU Bing-biao, ZHAO Ze-yu, AN Si-long, CHEN Hua-shuai, ZHAO Jian-nong

Hainan Medical University, Haikou, 570311, China

Keywords:Medulloblastoma Liquid biopsy of cerebrospinal fluid cfDNA

ABSTRACT

Medulloblastoma (MB) is an embryonic central nervous system malignancy, WHO class IV, which is common in children, occurs primarily in cerebellar vermicompos on the top of the IV ventricle,with a tendency to metastasize along the leptomeninge[1].With the in-depth study of molecular biology, the consensus divided MB into four molecular subtypes: WNT-activated, SHH-activated,Group 3, and Group 4, which the WHO updated in its diagnostic classification of MB in 2016, and the molecular subtypes are significantly associated with the prognosis[2,3].In the 2021 WHO Central Nervous System Tumor Classification, Group 3 and Group 4 in the MB molecular subtype have been merged into non-WNT/non-SHH, and under the research basis of large-scale methylation and transcriptome analysis, new subtypes have emerged in these three major molecular subtypes, which, like the three main molecular typings of MB, some of which are related to clinical pathology and genetic characteristics, have important diagnostic and prognostic value[4].At present, there is a comprehensive treatment regimen of surgery, radiotherapy and chemotherapy in MB, the postoperative radiotherapy and chemotherapy are critical treatment methods for MB, and the treatment decisions often need to be weighed between treatment benefits and toxic side effects, so it is important to monitor the treatment response[5].MRI and cerebrospinal fluid(CSF) cytology are commonly used methods to monitor response to treatment, but the information monitored has a certain lag,although tissue biopsy is the gold standard for disease diagnosis,invasive procedures are not easy to perform for intracranial lesions,liquid biopsy is a relatively noninvasive, easily accessible, and reproducible monitoring method[6].The discovery of tumor cells in lumbar puncture CSF in children with MB has long been reported,suggesting that CSF can be used as a pathway to obtain disease information for patients with MB[7].Recent studies in cancer patients have shown that it is feasible to extract cfDNA from body fluids to capture tumor-related genomic alterations, such as plasma,CSF, and urine[8].In MB patients, cfDNA was detected in the CSF samples taken before surgery, compared with 76.9% of cfDNA in plasma except for one patient with a very low variant allele frequencies (VAFs) and the detection rate was only 2.2%, indicating that cfDNA is abundant in CSF but is almost absent in plasma[9].The CSF cfDNA can reveal genomic alterations in tumors, facilitate molecular typing and risk stratification, and provide valuable information for diagnosis and prognosis.At the same time, the CSF cfDNA also detects genomic heterogeneity within tumors, which can identify small subclonals, and longitudinal analysis of the CSF cfDNA can understand the characteristics of genomic evolution when tiny residual diseases and tumors recur.Therefore, the CSF cfDNA analysis facilitates the clinical management of MB patients,helps design targeted treatment strategies, improves the therapeutic effect and avoids overtreatment[9].

1.Cell-free DNA

Cell-free DNA (cfDNA), also known as circulating tumor DNA (ctDNA), represents DNA fragments released during cellular turnover into biofluids, which can be detected by PCRbased technology or next-generation genome sequencing (NGS)technology[10].In some pathological conditions, such as trauma,infection, and cancer, cfDNA can be detected in relatively increased concentrations.Studies have found that the content of cfDNA in some patients with malignant tumors increases significantly, mainly due to the release of necrosis and apoptosis of tumor cells or adjacent tissues of tumors.cfDNA has been widely studied as a serum marker for extracranial malignancies such as bladder cancer, colorectal cancer, gastroesophageal cancer, ovarian cancer, pancreatic cancer,breast cancer, melanoma, hepatocellular carcinoma, and head and neck cancer, although its sensitivity to detection with current gene sequencing techniques in the serum of brain tumor patients is not high, studies have confirmed that cfDNA in CSF is more abundant than in serum[11].Current CSF biopsy is performed in brain tumors,such as glioma[12], medulloblastoma[13], central nervous system lymphoma[14] and brain metastases[15], all of which show good clinical application prospects.Therefore, the CSF cfDNA can be used as a liquid biopsy material to detect the characterization of malignancies of the central nervous system.

2.The clinical application of CSF cfDNA in MB

With the development of genome sequencing technology, the sensitivity of cfDNA detection derived from CSF samples has become higher and higher, and not only has the genome profile of cfDNA of the measured CSF been found to be highly consistent with the primary tumor tissue, but also richer than the primary tumor tissue[16].The CSF cfDNA is a marker of measurable residual disease (MRD), which refers to the small number of tumor cells left in the body after systematic treatment in tumor patients, which is the main cause of recurrence in MB patients[17].The CSF cfDNA testing is helpful for postoperative dynamic monitoring of tumor marker changes in MB patients, and can be used as the primary method for liquid biopsy of brain tumors to assist in diagnosis, risk stratification,assessment of treatment response, monitoring and prediction of recurrence[18].Therefore, the dynamic monitoring of CSF DNA in MB patients facilitates the clinical management of these patients.

2.1 Assists in clinical diagnosis

The determination of diagnosis, prognosis, and therapeutic targets for MB depends on the molecular signature of the tumor.At present,the molecular characterization of MB depends on the acquisition of tumor specimens, surgical excision is the main way to obtain specimens by tissue biopsy, stereotactic biopsy is also a common means of clinical diagnosis, but the implementation of cranial tumor surgery requires a multi-faceted consideration and is not easily implemented, even if the tumor tissue is obtained through surgery,due to the heterogeneity within the tumor, the information provided by the detected local tumor tissue about the molecular characteristics of the tumor may not be comprehensive[9].Genomic alterations in the CSF cfDNA were found to reproduce genomic alterations in tumors[6], so the analysis of the CSF cfDNA became a less invasive option for identifying MB genomic features.

MB occurs mainly in the cerebellum, and patients often have clinical manifestations of hydrocephalus, and they often require CSF drainage before surgery.CSF is available in a non-invasive manner,and the CSF cfDNA can be analyzed prior to surgery to provide information about the tumor[19].Therefore, the detection of the CSF cfDNA can provide surgeons and oncologists with important information about the disease to assess the balance between risk and benefit and to guide treatment strategies such as surgery, radiation therapy and chemotherapy.Studies have shown heterogeneity within MB tumor tissue, and the tumor genome is often altered during treatment[20].Therefore, the CSF liquid biopsy can provide a dynamic pattern of disease diagnosis and treatment.

2.2 Assist in risk stratification

In 2012, the pediatric neuro-oncology community reached an international consensus on MB molecule subtypes, reporting four different MB subtypes, namely, WNT-activated, SHH-activated,Group 3, and Group 4, which are important components of MB risk stratification[21].Patients under 16 years of age with MB who are WNT-activated generally have a better prognosis and a survival rate of ＞ 90% , The prognosis of adults with WNT-activated MB is unclear.Patients with MB with SHH-activated type exhibit prognosis associated with different age of diagnosis, histopathology,and specific underlying genetics, e.g., SHH-activated type can be subdivided into TP53 mutant subtypes and TP53 wild subtypes.The biological behavior of Group 3 and Group 4 is unclear.MYC amplification is a significant feature of Group 3 MB, occurs in about 15%-20% of patients and is associated with poor clinical prognosis.Chromatin changes are common in Group 4 MB, accounting for about 30%-40% of patients in total.Chromosome 17q abnormalities are common in both Group 3 and Group 4 types[22].The 2015 Heidelberg conference combined with MB molecular typing for risk stratification, which was divided into four groups according to the 5-year survival rate: low-risk group, standard-risk group, highrisk group and ultra-high-risk group[23], which shows that molecular characteristics are of great significance in the risk stratification of MB patients.

CfDNA-based CSF liquid biopsy reproduces molecular signatures within MB tumors.Escudero and colleagues[9] detected the key molecular alterations of MB in cfDNA, including TP53 and PTC1 mutations, MYCN and GLI2 amplification, SUFU and chromosome 17p deletions, whose characteristics were significantly associated with primary tumors, suggesting that the CSF cfDNA can provide rich information for MB molecular diagnosis and risk stratification.Liu and colleagues[17] have shown that the detection of baseline MRD is significantly associated with risk grouping, tumor metastatic status, molecular subgroup, and tumor location, all of which are specific manifestations of tumor risk stratification.Therefore, the CSF liquid biopsy can be used as an important supplement to MB risk stratification.DNA methylation is also applicable to the risklevel stratification of MB[24], Li and colleagues[25] analysis of DNA methylation of cfDNA in CSF between SHH and WNT subtypes of MB revealed significant differences between them, confirming that the DNA methylation status of the characteristic CpGs site of MB CSF can be used to identify the molecular subtype of MB.Therefore,cfDNA-based CSF liquid biopsy can assist in risk stratification in MB patients.

2.3 Assess response to treatment

Efficacy observation is the feedback on the implementation of treatment strategies, and the CSF liquid biopsy provides a pathway to assess response to treatment.The study found that the CSF cfDNA reproduced the subclonal genome profile of tumors, and the VAFs in tumors were significantly correlated with VAFs in CSF, suggesting that the CSF cfDNA can characterize the heterogeneity of MB within tumors, thereby helping to identify small invasive clones that promote drug resistance and thereby guiding changes in treatment strategies[9].Monitoring of cfDNA in the patient’s CSF enables the identification of tiny residual diseases and tumor evolution to assess tumor progression and response to treatment, thereby adjusting the intensity and duration of treatment to suit the specific state of the tumor at this time[17].In addition, in epigenetic studies, changes in DNA methylation levels were found at some CpGs sites during treatment, suggesting that the methylation levels of these CpGs can be used to assess response to treatment[24].In summary, The CSF liquid biopsy can assess treatment response by detecting changes in tumor genomic profiles and epigenetics.

2.4 Monitor for recurrence

Tumor recurrence is the leading cause of death in MB patients.Tumor recurrence and progression after systematic treatment are the most concerned issues of the patients and doctors, and the tissue biopsies are rarely performed when the tumor recurs.However, in the studies comparing genetic mutations in primary and recurrent tumors, it was found that other mutations may occur in recurrent tumors, that the genome of recurrent tumors may differ from those of primary tumors, and that the lack of tumor signature data may challenge the selection of effective second-line treatment strategies[9].Sun and colleagues[20] found that the level of the CSF cfDNA in MB patients was associated with tumor progression, leading to the idea that the CSF cfDNA could predict recurrence of the disease.Escudero and colleagues[9] analyzed the cfDNA of CSF in MB patients and found that tumor VAFs and copy number variations(CNVs) were highly consistent, combined with the follow-up results of patient imaging, it was believed that the CSF cfDNA monitoring could detect small residual diseases, so the CSF cfDNA could be used as a supplement to imaging and CSF cytology for early detection of tumor recurrence and guiding treatment strategies.Liu and colleagues[17] analyzed the cfDNA of CSF samples of MB patients with CNVs as markers of MRD, and found that all the CSF samples (n = 27) of 12 patients with tumor recurrence in imaging or cytology were positive for MRD; in patients with observed disease progression, 24/25 cases of CSF samples taken within 3 months were positive for MRD; in patients who did not observe disease progression, 193/209 CSF samples taken were MRD negative at the baseline; In the absence of imaging and/or cytological abnormalities,16/32 (50%) of MRD-positive patients suggested tumor recurrence during imaging follow-up after 3 months (median 8.3 months,intervals 3.0 to 29.4 months).In addition, tumor-related molecular profiles obtained from cfDNA found that 10q chromosome loss is a common change when MB patients relapse.Therefore, we believe that the CSF cfDNA has high sensitivity and specificity in MB monitoring for recurrence.

2.5 Predict prognosis

MB is a very malignant brain tumor, and people tend to pay attention to the prognosis of the disease.Knowing a patient’s diagnosis and prognosis information before surgery can help determine which patients have a good prognosis and can benefit from conservative tumor resection as well as mild doses of chemotherapy and radiation therapy.MRD is a risk-correcting factor and an indicator of short progression-free survival (PFS).The CSF cfDNA divide tumors into the correct molecular subtypes and risk subgroups and provides important information for prognosis.The level of cfDNA in CSF compared with tumor volume but no correlation was observed, while high levels of cfDNA were detected in preoperative CSF in patients with a very high risk of recurrence and disease progression was observed, and the abundance of CSF may be a valuable prognostic indicator[9].Finally, a study confirmed that Sun and colleagues[20] used a next-generation sequencing platform to deeply sequence CSF and only 15 of the 58 patients had cfDNA detected in CSF, and found that the detection of cfDNA was significantly related to tumor progression (P=0.003, Fisher’s exact test), therefore, the CSF liquid biopsy may provide useful prognostic information during the treatment and follow-up of MB.Subsequently, in a prospective study, Liu and colleagues[17] showed that, the CSF cfDNA of MB patients was continuously detected to evaluate MRD, and it was found that the PFS of patients with MRD positive was significantly lower than that of patients with MRD negative.Testing of MRD is independent of prognosis at the baseline(HR 1.36, 95% CI 0.67-2.76; Log rank P=0.4).Testing of MRD was significantly associated with PFS at the post-radiotherapy, the mid chemotherapy and the end-of-treatment (the post-radiotherapy:HR 6.53, 95% CI 3.22-13.26, Log rank P ＜ 0.000 1; the midchemotherapy: HR 6.35, 95% CI 3.06-13.19, log rank P ＜ 0.000 1; the end of treatment: HR 8.94, 95% CI 4.10-19.49, log rank P ＜0.000 1).In the mean risk group, MRD detected was significantly associated with PFS when it is at the post-radiotherapy, the midchemotherapy and the end-of- treatment(the post-radiotherapy:HR 49.63, 95% CI: 9.41 to 261.7; the mid-chemotherapy: HR:11.19, 95% CI: 3.45-36.32; the end-of-treatment: HR 11.43, 95%CI: 3.60-36.31).In the high-risk group, the detection of MRD was significantly associated with PFS at the end-of-treatment (HR 4.71,95% CI 1.56 to 14.21); The correlation between MRD detection and PFS tended to be significant at the post-radiotherapy and the middle of chemotherapy (the post-radiotherapy: HR 2.91, 95% CI 0.85 to 9.95; the mid-chemotherapy: HR 2.62, 95% CI 0.97 to 7.07).These findings suggest that MRD monitoring the CSF cfDNA assessment is associated with prognosis during treatment and provides some prognostic information.Therefore, the CSF liquid biopsy facilitates dynamic and real-time knowledge of the patient’s prognosis.

3.Changes in the genomic spectrum as tumors progress

By comparing the CSF cfDNA with the genome of tumor tissue,it is found that there are various common genetic features between the two, and it is also found that the gene mutations associated with the CSF cfDNA are more abundant than tumor tissue, indicating that CSF liquid biopsy has a more comprehensive genomic landscape than tissue biopsy[19].Gene sequencing of tumor tissue and CSF samples at MB recurrence revealed common mutations, including four predicted drivers:PTCH1:p.L1024Pfs, TP53:p.N239Tfs, CDKN2B: p.G124D,and KMT2A: p.W1199S.Mutations in PTCH1 and CDKN2B are validated by ddPCR in the DNA of tumors and normal brain tissue,as well as the cfDNA of CSF and plasma[20].Analysis of CNVs also revealed the presence of MYCN amplification, specific mutations associated with these primary tumors that provide information on MB resistance to SMO inhibitors at the time of recurrence.The CSF cfDNA sequencing also identified the tumor-specific mutation KMT2A:p.W1199S, which was validated by ddPCR.The author believes that these CSF-specific mutations may be missed in tumor biopsy analysis because of tumor heterogeneity.Despite limited levels of the cfDNA in CSF, mutations and associated genomic alterations found in primary tumors, including amplification of MYCN and GLI2, and partial loss of 17p were also detected[20].In addition, cfDNA analysis of CSF at the time of recurrence showed a genomic transformation whose molecular characteristics differed from primary or first-time relapsed tumors, when the levels of CSF cfDNA decreased, mutations in HCN1:p.Q828* were detected in recurrent primary tumors, but were not detected in the CSF cfDNA.Conversely, within 81 days of recurrence of disease, measured levels of ctDNA alterations in CSF were stable or elevated, and autopsy suggested ADD1:p.P718L VAFs: 41.6%-52% and ACTA2:p.Y190S VAFs: 13.2%-15%[20], suggesting that analysis of the CSF cfDNA could be used to monitor tumor genome evolution.Liu and colleagues[17] analysis of chromosomal aneuploidy in CSF-CNVs spectra suggests clonal selection or evolution in 12/15 (80%) of patients.Chromosome 10q loss is the loss of chromosome 10q when the tumor recurs, indicating that the loss of chromosome 10q is associated with tumor recurrence, and specific focal genetic changes such as SMAD2 and PIM1 amplification exist independently at the time of recurrence.In summary, we think the

CSF liquid biopsy can detect changes in the gene spectrum of tumors that differ from those of the primary tumor when they recur.Epigenetic alterations are also drivers of tumor recurrence.Li and colleagues[25] performed epigenetic analysis of cfDNA by CSF liquid biopsy found that the methylation levels of CpGs with different cfDNAs in CSF during the recurrence of disease in MB patients were higher or decreased than before recurrence, and the changes in cfDNA methylation in continuous CSF samples were related to tumor recurrence.Therefore, there are differences in the methylation of cfDNA in CSF when the tumor recurs.

4.Summary

In-depth studies of genomics and epigenetics have revolutionized the way tumors are treated.MB exhibits great spatial and temporal heterogeneity within and between tumors, CSF liquid biopsy can characterize and monitor MB, and the analysis of CNVs of cfDNA present in these samples can be used as a biomarker to determine the presence of MRD, helping predict the diagnosis, prognosis, and targeted treatment response of MB patients, enabling systematic monitoring, providing a pathway for the study of tumor resistance mechanisms, and promoting optimal treatment and clinical management of MB patients[13].The treatment of human cancer has shifted to the medical model of precision therapy, the choice of targeted therapy will depend on the genetic phenotype of individual patients, and cerebrospinal fluid biopsy is an important supplement for MB histological diagnosis and imaging diagnosis.Therefore,fluid biopsy based on CSFDNA can provide effective assistance for the clinical treatment of MB.