Cell free circulating tumor DNA in cerebrospinal fluid detects and monitors central nervous system involvement of B-cell lymphomas

Abstract

The levels of cell free circulating tumor DNA (ctDNA) in plasma correlated with treatment response and outcome in systemic lymphomas. Notably, in brain tumors, the levels of ctDNA in the cerebrospinal fluid (CSF) are higher than in plasma. Nevertheless, their role in central nervous system (CNS) lymphomas remains elusive. We evaluated the CSF and plasma from 19 patients: 6 restricted CNS lymphomas, 1 systemic and CNS lymphoma, and 12 systemic lymphomas. We performed whole exome sequencing or targeted sequencing to identify somatic mutations of the primary tumor, then variant-specific droplet digital PCR was designed for each mutation. At time of enrolment, we found ctDNA in the CSF of all patients with restricted CNS lymphoma but not in patients with systemic lymphoma without CNS involvement. Conversely, plasma ctDNA was detected in only 2/6 patients with restricted CNS lymphoma with lower variant allele frequencies than CSF ctDNA. Moreover, we detected CSF ctDNA in 1 patient with CNS lymphoma in complete remission and in 1 patient with systemic lymphoma, 3 and 8 months before CNS relapse was confirmed; indicating CSF ctDNA might detect CNS relapse earlier than conventional methods. Finally, in 2 cases with CNS lymphoma, CSF ctDNA was still detected after treatment even though a complete decrease in CSF tumor cells was observed by flow cytometry (FC), indicating CSF ctDNA better detected residual disease than FC. In conclusion, CSF ctDNA can better detect CNS lesions than plasma ctDNA and FC. In addition, CSF ctDNA predicted CNS relapse in CNS and systemic lymphomas.

Figure 1.

Genomic landscape of the cohort of study. (A) Pie chart representing the proportion of samples of each lymphoma type (see color legend). Two sectors in each sample depict whether the patient had systemic (gray) or central nervous system (CNS) restricted disease (black). (B) Circus plot representing all mutations identified across 16 lymphoma patients. A mutation was defined as driver according to Cancer Genome Interpreter (see Online Supplementary Appendix) colored in red (see Methods section). Genes with recurrent mutations have been highlighted in bold. (C) Bubble plot representing all driver mutations and droplet digital polymerase chain reaction (ddPCR) validated mutations identified in each patient. The bubble size represents the cancer allelic fraction (CAF) (see Online Supplementary Appendix) and the border has been highlighted in black if it has been found by ddPCR, in either the cerebrospinal fluid or plasma of each patient. NHL: non-Hodgkin lymphoma; DLBCL: diffuse large B-cell lymphoma; HGBCL: high-grade B-cell lymphoma; WM: Waldestrom macroglobulinemia; MCL: mantle cell lymphoma; BL: Burkitt lymphoma.

Figure 2.

Levels of circulating tumor DNA (ctDNA) correlate with disease progression and response to treatment in Bcell lymphomas. (A-F) Longitudinal monitoring of two patients with central nervous system (CNS) lymphomas (NHL2 and NHL4) and (G-L) six patients with systemic lymphomas with no evidence of CNS disease (NHL8, NHL9, NHL10, NHL13, NHL18, and NHL19) through (A) cerebrospinal fluid (CSF) and (D, G and J) plasma ctDNA analysis (variant allele frequency [VAF] values for each mutation detected using droplet digital polymerase chain reaction [ddPCR]) and its correlation with (B) CSF flow cytometry (cells/μL), (C) cytology (+/- presence/absence of cells), and (F, I and L) radiological imaging (magnetic resonance imaging [MRI] and positron emission tomography/computed tomography scans). Data points are vertically aligned (E, H and K). Clinical information for each patient includes type of treatment and radiological response assessment. MRI scans for patient NHL4 are not shown given that the patient only presented leptomeningeal involvement. Dx: time of diagnosis; IT MTX: intrathecal methotrexate; R-CHOP: combined therapy (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisolone); Burk: Burkimab regimen; CR: complete response; EOT: end of treatment; m: month; w: week.

Figure 3.

Cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) is more sensitive than flow cytometry (FC) to detect central nervous system (CNS) relapse and residual disease in CNS-restricted lymphomas. Longitudinal monitoring of three patients with CNS-restricted lymphomas (NHL3, NHL5 and NHL6) through (A) CSF and (D) plasma ctDNA analysis (variant allele frequency [VAF] values for each mutation obtained by droplet digital polymerase chain reaction [ddPCR]) and its correlation with (B) CSF flow cytometry (cells/L), (C) cytology (+/- presence/absence of cells). (E) Type of treatment and response assessment, (F) radiological imaging (MRI). Data points are vertically aligned. MRI scans for patient NHL6 and NHL5 are not shown given that the patient only presented leptomeningeal involvement at diagnosis. Dx: time of diagnosis; IT MTX: intrathecal methotrexate; BAM-R: combined therapy (rituximab, carmustine, cytarabine and methotrexate); ASCT: autologous stem cell transplant; CR: complete response; LM: leptomeningeal; EOT: end of treatment; d: day; m: month; NA: not available. *CSF sample was not available for ctDNA analyses.

Figure 4.

Cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) detects central nervous system (CNS) involvement and residual disease in patients with systemic lymphoma and is more sensitive than conventional tests. Longitudinal analyses of (A) CSF and (D) plasma ctDNA (VAF values for each mutation detected using ddPCR) and correlation with (B) CSF FC (cells/L), (C) cytology (+/- presence/absence of cells), and (E) type of treatment and radiological response assessment. (F) Radiological imaging (magnetic resonance imaging [MRI] and positron emission tomography/computed tomography scans) in two patients with systemic lymphoma (NHL16 and NHL17) that present CNS relapse. Data points are vertically aligned. MRI scans for patient NHL17 are not shown given that the patient only presented leptomeningeal involvement. Dx: time of diagnosis; R-CHOP: combined therapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone); IT MTX: intrathecal methotrexate; CR: complete response; P: parenchymal; LM: leptomeningeal; EOT: end of treatment; m: month; d: days; NA: not available. *CSF sample was not available for ctDNA analyses