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S. Bobillo et al.
nial magnetic resonance imaging (MRI) along with brain excisional biopsy or analysis of cerebrospinal fluid (CSF) or vitreous fluid.4 In some cases, biopsies can be challenging owing to the difficulty in accessing the tumor and the inher- ent risks associated with cranial surgery.
Patients with aggressive systemic lymphomas and risk factors for CNS relapse are routinely monitored by cytol- ogy and flow cytometry (FC) analysis of CSF. Cytology is highly specific but has a very limited sensitivity, showing up to 40% false negatives. In contrast, FC is more sensitive, detecting malignant cells in up to 5-15% of patients with negative cytology.5,6 However, FC still has some limitations, and in a relevant fraction of patients, recurrence in the CNS is found shortly after a negative result by FC, suggesting that tumor cells were undetected.
Several studies have demonstrated that cell free circulat- ing tumor DNA (ctDNA) can be detected in plasma from patients with B-cell lymphomas and correlates with meta- bolic tumor volume and outcome.7,8 Due to the difficulties in the diagnosis of brain tumors, there is growing interest in the analysis of ctDNA in plasma and CSF and their poten- tial utility in the management of these patients. Thus, we and others have explored the analysis of CSF in solid brain tumors as a better source of ctDNA compared with plasma.9-12 As per B-cell lymphomas, only a minority of patients with restricted CNS lymphomas present detectable ctDNA in plasma13,14 and only a few studies of ctDNA in CSF have been performed so far.15-19 In this regard, the MYD88 L265P mutation has been identified in the CSF ctDNA from some patients with PCNSL and lymphoplas- macytic lymphoma with CNS involvement.17-19 More recently, a different study using a next-generation sequenc- ing (NGS)-based assay, detected at least one tumor-derived genetic alteration in the CSF from eight patients with relapsed CNS lymphoma and demonstrated that CSF ctDNA levels correlated with treatment response.15
Taken together, these studies demonstrate that CSF ctDNA could be detected in patients with CNS lymphomas and could be useful to monitor the disease, but a thorough analysis of its relevance on the diagnosis and monitoring of the disease is still needed.
Here, we conducted a study to explore whether the analysis of ctDNA in the CSF and plasma could be useful to complement the diagnosis and molecular profile of tumors, as well as to monitor treatment response in CNS lym- phomas. In addition, we evaluated whether the presence of CSF ctDNA in patients with systemic lymphoma could be more sensitive to detect CNS malignancies than CSF stan- dard analyses and plasma ctDNA.
Methods
Patients and samples
Nineteen patients diagnosed with the following conditions were included: restricted CNS lymphomas, n=6; PCNSL, n=1; SCNSL, n=5; systemic lymphoma with concomitant CNS involve- ment, n=1; and systemic lymphoma without CNS disease but risk factors for CNS relapse, n=12. Risk factors for CNS relapse were defined as: diffuse large B-cell lymphoma (DLBCL) with testis, kidney or adrenal involvement; DLBCL with involvement of two or more extranodal sites along with high lactate dehydrogenase; high-grade B-cell lymphoma (HGBCL) with MYC and/or BCL2 rearrangements or Burkitt lymphoma. All patients were diagnosed and treated at Vall d’Hebron University Hospital (Barcelona,
Spain). Diagnosis of CNS disease was established by MRI imaging and tumor biopsy in cases with parenchymal involvement, or by FC and cytology in cases with leptomeningeal disease.
A written informed consent was obtained from all individuals in accordance with the Declaration of Helsinki and the study was approved by the local clinical research ethics committee. As part of standard of care practice, in patients with systemic lymphoma and risk factors for CNS relapse, prophylactic intrathecal (IT) methotrexate (MTX) was administered every 3 weeks together with systemic chemotherapy. In addition, in cases with lep- tomeningeal disease, IT chemotherapy was administrated every 3-4 days until malignant cells were not detected by FC. CSF (1-2 mL) and plasma were collected before treatment in all patients and sequentially in patients who received IT chemotherapy as part of routine practice. Cytology and FC were performed in all CSF sam- ples. Two out of 7 patients with CNS lymphoma and 3 out of 12 with systemic lymphoma received steroids before the baseline CSF sample. Evaluation of response was assessed at the end of treatment and/or on suspicion of disease progression by using MRI imaging in cases with CNS parenchymal involvement and by CSF analysis (FC and cytology) in patients with leptomeningeal disease. Positron emission tomography/computed tomography (PET/CT) was used to assess response at the end of treatment in patients with systemic lymphoma.
Sample collection and DNA extraction
The median volume of plasma and CSF obtained was 2.5 mL (range: 1-6) and 1 mL (range: 0.3-2), respectively. Peripheral blood was collected in tubes containing K2EDTA (Vacutainer) and cen- trifuged at 1600xg for 10 minutes (min). The plasma was then transferred to another tube that was further centrifuged at 3000xg for 5 min. CSF samples were centrifuged at 3000xg for 5 min. The supernatant was collected and CSF-derived and plasma-derived circulating cell free DNA (cfDNA) was extracted using the QIAamp Circulating Nucleid Acids kit and quantified using a flu- orimeter. DNA was extracted from formalin-fixed paraffin- embedded tumor samples using the QIAamp DNA FFPE tissue kit. Germline DNA was extracted from peripheral blood granulocytes using the Qiagen DNeasy Blood and Tissue kit.
DNA sequencing and mutation genomic analysis
We performed DNA sequencing of the tumors of 16 out of 19 patients: 11 of 16 were sequenced using a 300 gene targeted NGS panel (Vall d’Hebron Institute of Oncology, Genomics Facility) and in the remaining five, whole-exome DNA sequencing (WES) was performed in both the tumor and germline DNA. SureSelect Human All Exon V5 (Agilent Technologies) was used to perform whole exome enrichment for the Illumina paired-read sequencing platform HiSeq2500 with a read length of 2x100bp (Online Supplementary Appendix). The amount of DNA used for NGS panel and WES was 1 mg and 0.4-4 mg, respectively. In 3 out of 19 patients (NHL1, NHL4 and NHL5), MYD88 L265P mutation was detected in the tumor by routine genomic analysis performed at diagnosis, therefore tumor DNA sequencing was not performed.
Droplet digital polymerase chain reaction and quantification of circulating tumor-specific DNA
Given the limited amount of cfDNA available, ddPCR was per- formed to determine the presence of ctDNA mutations in the CSF and plasma, ensuring sensitivity and precision. A set of driver mutations along with additional mutations not predicted as driv- ers by Cancer Genome Interpreter (CGI), but relevant according to expert knowledge, were selected for further validation by ddPCR.
Custom Taqman SNP genotyping assays for ddPCR were designed to specifically detect the selected point mutations and
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