Non-Hodgkin Lymphoma ARTICLE Cell free circulating tumor DNA in cerebrospinal Ferrata Storti Foundation fluid detects and monitors central nervous system involvement of B-cell lymphomas Sabela Bobillo,1* Marta Crespo,1* Laura Escudero,2* Regina Mayor,2 Priyanka Raheja,1 Cecilia Carpio,1 Carlota Rubio-Perez,2 Bárbara Tazón-Vega,1Carlos Palacio,1 Júlia Carabia,1 Isabel Jiménez,1 Juan. C. Nieto,1 Julia Montoro,1 Francisco Martínez-Ricarte,3 Josep Castellví,4 Marc Simó,5 Lluis Puigdefàbregas,1 Pau Abrisqueta,1 Francesc Bosch1# and Joan Seoane2,6# Haematologica 2021 1 Laboratory of Experimental Hematology, Department of Hematology, Vall d'Hebron Volume 106(2):513-521 Institute of Oncology (VHIO), Vall d’Hebron University Hospital (HUVH), Universitat Autònoma de Barcelona, Department of Medicine; 2Translational Research Program, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron University Hospital (HUVH), Universitat Autònoma de Barcelona; 3Department of Neurosurgery, Universitat Autònoma de Barcelona, Hospital Vall d'Hebron; 4Department of Pathology, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona; 5Department of Nuclear Medicine, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona and 6Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain and CIBERONC *SB, MC and LE contributed equally as co-first authors. FB and JS contributed equally as co-senior authors. # ABSTRACT T he levels of cell free circulating tumor DNA (ctDNA) in plasma cor- relate with treatment response and outcome in systemic lym- phomas. Notably, in brain tumors, the levels of ctDNA in the cere- brospinal 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 sys- temic and CNS lymphoma, and 12 systemic lymphomas. We performed whole exome sequencing or targeted sequencing to identify somatic muta- Correspondence: tions of the primary tumor, then variant-specific droplet digital polymerase chain reaction was designed for each mutation. At time of enrollment, we JOAN SEOANE jseoane@vhio.net found ctDNA in the CSF of all patients with restricted CNS lymphoma but not in patients with systemic lymphoma without CNS involvement. FRANCESC BOSCH Conversely, plasma ctDNA was detected in only 2 out of 6 patients with fbosch@vhio.net restricted CNS lymphoma with lower variant allele frequencies than CSF Received: October 22, 2019. ctDNA. Moreover, we detected CSF ctDNA in one patient with CNS lym- phoma in complete remission and in one patient with systemic lymphoma, Accepted: February 18, 2020. 3 and 8 months before CNS relapse was confirmed, indicating that CSF Pre-published: February 20, 2020. ctDNA might detect CNS relapse earlier than conventional methods. Finally, in two cases with CNS lymphoma, CSF ctDNA was still detected https://doi.org/10.3324/haematol.2019.241208 after treatment even though no tumoral cells were observed by flow cytometry (FC), indicating that CSF ctDNA detected residual disease better than FC. In conclusion, CSF ctDNA can detect CNS lesions better than plas- ©2021 Ferrata Storti Foundation ma ctDNA and FC. In addition, CSF ctDNA predicted CNS relapse in CNS Material published in Haematologica is covered by copyright. and systemic lymphomas. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Introduction Copies of published material are allowed for personal or inter- nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: Central nervous system (CNS) involvement in B-cell malignancies is associated https://creativecommons.org/licenses/by-nc/4.0/legalcode, with dismal prognosis, especially in the relapse setting.1 Primary central nervous sys- sect. 3. Reproducing and sharing published material for com- tem lymphoma (PCNSL) is defined by the presence of CNS lymphoma in the mercial purposes is not allowed without permission in writing absence of systemic disease.2 Conversely, secondary involvement of the CNS from the publisher. (SCNSL) is characterized by CNS infiltration with concomitant or previous history of systemic lymphoma.3 The diagnosis of CNS lymphoma is commonly based on cra- haematologica | 2021; 106(2) 513 S. Bobillo et al. nial magnetic resonance imaging (MRI) along with brain Spain). Diagnosis of CNS disease was established by MRI imaging excisional biopsy or analysis of cerebrospinal fluid (CSF) or and tumor biopsy in cases with parenchymal involvement, or by vitreous fluid.4 In some cases, biopsies can be challenging FC and cytology in cases with leptomeningeal disease. owing to the difficulty in accessing the tumor and the inher- A written informed consent was obtained from all individuals ent risks associated with cranial surgery. in accordance with the Declaration of Helsinki and the study was Patients with aggressive systemic lymphomas and risk approved by the local clinical research ethics committee. As part factors for CNS relapse are routinely monitored by cytol- of standard of care practice, in patients with systemic lymphoma ogy and flow cytometry (FC) analysis of CSF. Cytology is and risk factors for CNS relapse, prophylactic intrathecal (IT) highly specific but has a very limited sensitivity, showing methotrexate (MTX) was administered every 3 weeks together up to 40% false negatives. In contrast, FC is more sensitive, with systemic chemotherapy. In addition, in cases with lep- detecting malignant cells in up to 5-15% of patients with tomeningeal disease, IT chemotherapy was administrated every negative cytology.5,6 However, FC still has some limitations, 3-4 days until malignant cells were not detected by FC. CSF (1-2 and in a relevant fraction of patients, recurrence in the CNS mL) and plasma were collected before treatment in all patients and is found shortly after a negative result by FC, suggesting sequentially in patients who received IT chemotherapy as part of that tumor cells were undetected. routine practice. Cytology and FC were performed in all CSF sam- Several studies have demonstrated that cell free circulat- ples. Two out of 7 patients with CNS lymphoma and 3 out of 12 ing tumor DNA (ctDNA) can be detected in plasma from with systemic lymphoma received steroids before the baseline patients with B-cell lymphomas and correlates with meta- CSF sample. Evaluation of response was assessed at the end of bolic tumor volume and outcome.7,8 Due to the difficulties treatment and/or on suspicion of disease progression by using in the diagnosis of brain tumors, there is growing interest in MRI imaging in cases with CNS parenchymal involvement and by the analysis of ctDNA in plasma and CSF and their poten- CSF analysis (FC and cytology) in patients with leptomeningeal tial utility in the management of these patients. Thus, we disease. Positron emission tomography/computed tomography and others have explored the analysis of CSF in solid brain (PET/CT) was used to assess response at the end of treatment in tumors as a better source of ctDNA compared with patients with systemic lymphoma. plasma.9-12 As per B-cell lymphomas, only a minority of patients with restricted CNS lymphomas present detectable Sample collection and DNA extraction ctDNA in plasma13,14 and only a few studies of ctDNA in The median volume of plasma and CSF obtained was 2.5 mL CSF have been performed so far.15-19 In this regard, the (range: 1-6) and 1 mL (range: 0.3-2), respectively. Peripheral blood MYD88 L265P mutation has been identified in the CSF was collected in tubes containing K2EDTA (Vacutainer) and cen- ctDNA from some patients with PCNSL and lymphoplas- trifuged at 1600xg for 10 minutes (min). The plasma was then macytic lymphoma with CNS involvement.17-19 More transferred to another tube that was further centrifuged at 3000xg recently, a different study using a next-generation sequenc- for 5 min. CSF samples were centrifuged at 3000xg for 5 min. The ing (NGS)-based assay, detected at least one tumor-derived supernatant was collected and CSF-derived and plasma-derived genetic alteration in the CSF from eight patients with circulating cell free DNA (cfDNA) was extracted using the relapsed CNS lymphoma and demonstrated that CSF QIAamp Circulating Nucleid Acids kit and quantified using a flu- ctDNA levels correlated with treatment response.15 orimeter. DNA was extracted from formalin-fixed paraffin- Taken together, these studies demonstrate that CSF embedded tumor samples using the QIAamp DNA FFPE tissue kit. ctDNA could be detected in patients with CNS lymphomas Germline DNA was extracted from peripheral blood granulocytes and could be useful to monitor the disease, but a thorough using the Qiagen DNeasy Blood and Tissue kit. analysis of its relevance on the diagnosis and monitoring of the disease is still needed. DNA sequencing and mutation genomic analysis Here, we conducted a study to explore whether the We performed DNA sequencing of the tumors of 16 out of 19 analysis of ctDNA in the CSF and plasma could be useful to patients: 11 of 16 were sequenced using a 300 gene targeted NGS complement the diagnosis and molecular profile of tumors, panel (Vall d’Hebron Institute of Oncology, Genomics Facility) and as well as to monitor treatment response in CNS lym- in the remaining five, whole-exome DNA sequencing (WES) was phomas. In addition, we evaluated whether the presence of performed in both the tumor and germline DNA. SureSelect CSF ctDNA in patients with systemic lymphoma could be Human All Exon V5 (Agilent Technologies) was used to perform more sensitive to detect CNS malignancies than CSF stan- whole exome enrichment for the Illumina paired-read sequencing dard analyses and plasma ctDNA. 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 Methods patients (NHL1, NHL4 and NHL5), MYD88 L265P mutation was detected in the tumor by routine genomic analysis performed at Patients and samples diagnosis, therefore tumor DNA sequencing was not performed. Nineteen patients diagnosed with the following conditions were included: restricted CNS lymphomas, n=6; PCNSL, n=1; Droplet digital polymerase chain reaction and SCNSL, n=5; systemic lymphoma with concomitant CNS involve- quantification of circulating tumor-specific DNA ment, n=1; and systemic lymphoma without CNS disease but risk Given the limited amount of cfDNA available, ddPCR was per- factors for CNS relapse, n=12. Risk factors for CNS relapse were formed to determine the presence of ctDNA mutations in the CSF defined as: diffuse large B-cell lymphoma (DLBCL) with testis, and plasma, ensuring sensitivity and precision. A set of driver kidney or adrenal involvement; DLBCL with involvement of two mutations along with additional mutations not predicted as driv- or more extranodal sites along with high lactate dehydrogenase; ers by Cancer Genome Interpreter (CGI), but relevant according to high-grade B-cell lymphoma (HGBCL) with MYC and/or BCL2 expert knowledge, were selected for further validation by ddPCR. rearrangements or Burkitt lymphoma. All patients were diagnosed Custom Taqman SNP genotyping assays for ddPCR were and treated at Vall d’Hebron University Hospital (Barcelona, designed to specifically detect the selected point mutations and 514 haematologica | 2021; 106(2) ctDNA in cerebrospinal fluid of patients with CNS lymphoma A B C 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: Waldeström macroglobulinemia; MCL: mantle cell lymphoma; BL: Burkitt lymphoma. the corresponding wild-type alleles. Genomic DNA from tumor CNS involvement. CSF was obtained at the same time as tissues (10 ng), germline DNA from peripheral blood granulocytes plasma in all patients. WES or targeted sequencing of the (10 ng), plasma DNA, and CSF DNA (1-5 ng) were used for ddPCR tumors was performed to identify somatic mutations analysis using the QX200 Droplet Digital PCR system according (Figure 1). Variant-specific ddPCR detecting 1-5 variants per to manufacturer's protocols and the literature.20 patient was performed to detect ctDNA. To demonstrate that the variant-specific ddPCR was highly specific, we performed MYD88 L265P ddPCR in ten Results CSF samples obtained from patients with hydrocephalus without a brain tumor (n=6), and from patients diagnosed Circulating tumor DNA in the cerebrospinal fluid of with glioma (n=3) and a cavernoma (n=1). No mutant allele patients with central nervous system restricted B-cell was found in any of the cases. lymphoma is more abundant than in plasma Analysis of the ctDNA in the CSF and plasma of the six Here, we sought to analyze the presence of ctDNA in the CNS restricted lymphomas showed detectable ctDNA at CSF and plasma from 19 patients with B-cell lymphomas high variant allele frequency (VAF) (ranging from 1% to with and without CNS disease at time of enrollment (Table 95%) in all cases. In contrast, ctDNA in plasma was only 1). Six patients exhibited CNS restricted disease, one sys- detected in 2 out of 6 cases at very low VAF (always <5%) temic and CNS disease, and 12 systemic disease with no (Table 1), highlighting that in CNS restricted lymphomas, haematologica | 2021; 106(2) 515 S. Bobillo et al. Table 1. Patients’ characteristics and tumor mutations at the time of enrolment and outcome. ID Age Histol Dx Disease Systemic CNS CNS Tumor Tumor VAF VAF VAF Outcome status inv inv (FC/C) seq mutations tumor CSF plasma CNS lymphoma NHL1 59 DLBCL PCNSL New dx No P -/- - MYD88 L265P - 48% N CR NHL2 60 DLBCL SCNSL Relapse No P, LM +/- WES CD79B Y197D 17% 9% - CR MYD88 L265P 22% 25% - FOXO1 D69A 13% - - TMSB4A Q24P 54% 20% - NHL3 53 DLBCL SCNSL Relapse No P, LM +/+ Panel B2M M1T 41% N N PD, died BCOR N145S 68% 52% N CREBBP R1664C 21% N N NHL4 75 WM SCNSL Relapse No LM +/+ - MYD88 L265P - 33% N CR NHL5 58 DLBCL SCNSL Relapse No P, LM +/+ - MYD88 L265P 86% 29% 5% CR, CNS relapse NHL6 73 MCL SCNSL Relapse No LM +/+ Panel ATM H2872L 31% 34% 2% PD, died MEF2B K23R 90% 95% 4% CNS and systemic lymphoma NHL7 72 MCL SCNSL Relapse LN LM +/+ Panel BRAF N581S 44% N 7% PD, died Systemic lymphoma NHL8 50 HGBCL - New dx LN, No -/- WES MYC Y27C 28% N 16% CR bone, ARID1A E1104 32% N 20% intestine MEF2B D83G 33% N 18% MLL2 C533fs 50% N 31% CCND3 P284fs 29% N 16% NHL9 38 HGBCL - New dx LN, No -/- WES ID3 Y48* 38% N 12% CR intestine DDX3X Q360fs 98% N 15% NHL10 73 DLBCL - New dx LN, No -/- Panel MLL2 splicing 60% N 19% CR kidney MLL2 P3139A 34% N 12% NHL11 72 DLBCL - New dx LN, lung, No -/- Panel TP53 R273C 32% N 12% CR ileum CREBBP Y145C 25% N 27% NHL12 86 DLBCL - New dx LN, breast No -/- Panel MCL1 L186F 31% N 0.9% CR PTNPN6 T80fs 26% N N NHL13 77 DLBCL - New dx LN, No -/- WES MYD88 L265P 29% N 5% CR testes PIM1 L2F 27% N 3% MEF2B E77K 27% N 5% NHL14 62 DLBCL - New dx Teste No -/- Panel HSD3B2 L108W 22% N N CR MYD88 L265P 31% N N NHL15 75 DLBCL - New dx LN, testes, No -/- Panel BCOR S1517fs 63% N N CR liver, adrenal EZH2 P557L 34% N N NHL16† 57 DLBCL - New dx LN, sinus, No -/- Panel MYD88 V217F 28% N N CR, CNS relapse bones ASXL2 K927R 42% N N NHL17 58 HGBCL - New dx LN No -/- Panel CREBBP S1436I 50% N 16% CR, CNS relapse NHL18† 60 BL - New dx LN, No -/- WES ID3 L54V 37% N N CR intestine KRAS G12V 34% N 1% TCF3 D561E 42% N N ABL1 G321R 43% N 0.5% NHL19† 65 BL - New dx LN, bone No -/- Panel ARID1A del 9% N 1% CR BL: Burkitt lymphoma; C: cytology; CNS: central nervous system; CR: complete response; CSF: cerebrospinal fluid; DLBCL: diffuse large B-cell lymphoma; Dx: diagnosis; FC: flow cytometry; HGBCL: high-grade B-cell lymphoma; Histol: histology; ID: patient identification number; inv: involvement; LM: leptomeningeal; LN: lymph nodes; MCL: mantle cell lymphoma; N: not detectable; Neg: negative; NHL: non-Hodgkin lymphoma; P: parenchyma; PCNSL: primary central nervous system lymphoma; PD: progressive disease; Pos: pos- itive; SCNSL: secondary central nervous system lymphoma; seq: sequencing; VAF: variant allele frequency; WES: whole exome sequencing; WM: Waldeström macroglobulinemia. † Plasma samples were collected after administration of systemic chemotherapy. 516 haematologica | 2021; 106(2) ctDNA in cerebrospinal fluid of patients with CNS lymphoma Figure 2. Levels of circulating tumor DNA (ctDNA) correlate A with disease progression and response to treatment in B- cell lymphomas. (A-F) Longitudinal monitoring of two patients with central nervous system (CNS) lymphomas (NHL2 and NHL4) and (G-L) six patients with systemic lym- phomas with no evidence of CNS disease (NHL8, NHL9, NHL10, NHL13, NHL18, and NHL19) through (A) cere- brospinal 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 imag- ing [MRI] and positron emission tomography/computed B 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: com- bined therapy (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisolone); Burk: Burkimab regimen; CR: complete response; EOT: end of C treatment; m: month; w: week. D F E G H I J K L haematologica | 2021; 106(2) 517 S. Bobillo et al. A B C D E F 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 corre- lation with (B) CSF flow cytometry (cells/mL), (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. ctDNA is better identified in the CSF than in plasma. ed lymphoma cells. In one case (NHL2), cytology was nor- Interestingly, these two cases with restricted CNS lym- mal but a small amount of tumor cells was observed by FC phoma in which plasma ctDNA was detected had a previ- (0.06 cell/mL). Notably, all six cases exhibited CSF ctDNA. ous history of systemic disease. This finding might reveal a Importantly, in a patient with newly diagnosed PCNSL systemic nature of some restricted SCNSL and deserves fur- (NHL1) we could not detect lymphoma cells by FC or cytol- ther investigation in larger studies. ogy; however, ctDNA was abundantly found in the CSF We then analyzed 12 patients with systemic lymphoma (Table 1). These results indicate that ctDNA analysis of the without CNS involvement but with high risk for CNS CSF can improve the detection of CNS lymphoma by con- relapse. In these cases, lumbar puncture was performed to ventional techniques. rule out CNS infiltration and to administer IT MTX. As opposed to cases with CNS involvement, no ctDNA was Cerebrospinal fluid circulating tumor DNA can be used detected in the CSF while it was detected at high VAF in the to monitor central nervous system tumor burden and plasma of the majority of cases (Table 1). Finally, the patient response to treatment with systemic and concomitant CNS disease had ctDNA in To address whether the amount of CSF ctDNA could be plasma but not in the CSF, suggesting that some CNS useful to monitor tumor burden in CNS lymphomas, we lesions cannot be captured through CSF ctDNA. analyzed sequential concomitant samples of CSF and plas- ma. CSF was obtained as part of the standard procedures Central nervous system circulating tumor DNA exhibits before each administration of IT MTX or on suspicion of higher sensitivity than flow cytometry in detecting relapse. Tumor burden was evaluated by MRI and/or FC central nervous system lesions depending on the lymphoma location. We observed that We next correlated the detection of ctDNA with the iden- CSF ctDNA levels correlated with disease response and tification of malignant cells by conventional tests in the progression (Figure 2). In addition, tumor burden measured same CSF samples at time of enrollment. Cytology detected by MRI and FC was concordant with CSF ctDNA levels in the presence of malignant cells in 4 out of 6 CNS restricted two patients with CNS lymphoma (Figure 2). Importantly, lymphomas. In these four cases the FC analysis also detect- plasma ctDNA levels remained constantly low (VAF <6%) 518 haematologica | 2021; 106(2) ctDNA in cerebrospinal fluid of patients with CNS lymphoma A B C D E F 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/mL), (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 in sequential samples and did not correlate with the disease the CSF could be more sensitive than conventional CSF status, underlining the fact that plasma ctDNA is not useful analyses to monitor CNS lymphoma and detect CNS to monitor the activity of CNS restricted lymphomas relapse. (Figure 2). In two cases of CNS lymphoma (NHL3 and NHL5), we Six patients with systemic lymphoma without CNS observed that CSF ctDNA levels increased earlier than the involvement had sequential plasma samples collected at the detection of cells by FC. In NHL3, after the first dose of IT same time as CSF. We observed that plasma ctDNA became MTX, despite the fact that the patient did not exhibit a undetectable after the first cycle of treatment in all cases, in complete neurological response, we observed a sharp agreement with PET/CT results (Figure 2), suggesting that decrease in CSF ctDNA levels concomitant with a decrease plasma ctDNA is a good tool to monitor response to treat- in the tumor cells by FC. However, after three doses of IT ment in these patients. MTX, although FC was negative, a clear ctDNA increase was observed. The patient died 1 month later due to pro- Cerebrospinal fluid circulating tumor DNA can identify gression (Figure 3). In NHL5, CSF ctDNA levels also central nervous system relapse and residual central decreased with response to treatment and remained unde- nervous system disease better than flow cytometry tectable after complete remission. However, CSF ctDNA We then addressed whether the presence of ctDNA in reappeared in two CSF samples collected 2 and 4 months haematologica | 2021; 106(2) 519 S. Bobillo et al. after treatment due to the presence of minimal neurological and cytology. Regrettably, around 50-60% of patients symptoms, when brain-spinal MRI and CSF FC did not with CNS lymphoma will eventually relapse.21 Notably, show any evidence of disease; 8 months later CNS relapse we observed that CSF ctDNA was able to detect CNS was confirmed (Figure 3). Taken together, our results indi- lesions better than FC and cytology. In addition, CSF cate that ctDNA can be a more sensitive tool than FC to ctDNA was able to predict CNS relapse months before monitor relapse of CNS lymphomas. MRI or FC, indicating that it can be used for early detec- In one additional case of CNS lymphoma (NHL6) treat- tion of tumor relapse. ment with IT MTX decreased the number of cells in the CNS relapse from systemic lymphoma is a fatal compli- CSF till they were no longer detectable by FC. However, cation with an overall survival of less than 6 months, and CSF ctDNA was still detectable. Notably, this patient early identification of patients with CNS relapse is of para- relapsed shortly after the analysis (Figure 3). In a similar mount value.3 High-risk patients are usually tested for CNS way, we found persistence of ctDNA in the CSF from case disease by CSF analysis including cytology and FC. NHL17 after treatment for CNS relapse, despite the fact Although FC is a more sensitive test than conventional that FC did not detect malignant cells. Four months later, cytology,5,6 it has some limitations22 and some patients with the patient relapsed. (Figure 4). These results point out no evidence of CNS disease by FC still relapse, suggesting that the detection of CSF ctDNA may help to identify the presence of undetected malignant cells in the CSF. We patients with residual disease who are likely to relapse. identified ctDNA in the CSF in the absence of MRI or FC A subset of patients with systemic lymphoma presents detection earlier than CNS relapse. This indicates that CSF high risk of CNS relapse, which frequently occurs during ctDNA could facilitate early detection of CNS relapse in treatment or shortly thereafter. To address whether the high-risk systemic lymphomas who undergo serial lumbar presence of CSF ctDNA could be more sensitive than FC punctures to administrate prophylactic IT chemotherapy, to detect CNS disease, we analyzed sequential CSF sam- therefore improving management of these patients. ples from eight patients with systemic lymphoma Moreover, we observed the persistence of high levels of obtained while receiving prophylactic IT MTX. Two out CSF ctDNA in some patients even though FC did not of 8 patients relapsed in the CNS during the time of study detect tumor cells. This suggested that the detection of a (Figure 4). In one case (NHL17), we found CSF ctDNA at residual disease by CSF ctDNA could be used to deter- the same moment in which FC showed a positive result. mine the type and duration of treatments. However, we could not detect ctDNA in the CSF samples Taken together, and with the limitation of the reduced collected 9 and 7 months before CNS progression. number of patients analyzed, our findings show that CSF Notably, in case NHL16, the detection of ctDNA in the ctDNA can be an important tool to complement standard CSF with a VAF of 7% antedated by 3 months the CNS procedures to evaluate the CNS lymphoma disease sta- relapse. Importantly, at the time of ctDNA detection, the tus. This technology can be exploited as a 'liquid biopsy' patient was asymptomatic and CSF FC was normal of CNS lymphoma opening a novel way forward for (Figure 4). This case points out that CSF ctDNA could research in circulating biomarkers of CNS lymphoma detect CNS involvement in systemic lymphomas earlier with an important impact on the future characterization, than standard procedures. Finally, in the remaining six diagnosis, prognosis, and management of this type of dis- patients with systemic lymphoma and sequential CSF eases. samples, ctDNA was not detected and, with a median fol- low-up of 18 months (range: 12-28 months), these patients Disclosures did not develop CNS disease. JS is a co-founder of Mosaic Biomedicals and Northern Biologics. JS received grant/research support from Mosaic Biomedicals, Northern Biologics and Roche/Glycart. M. C. has Discussion received research funding from Karyopharm, Pharmacyclics, Roche, Arqule and AstraZeneca. Francesc Bosch has received The diagnosis and monitoring of CNS lymphoma are research funding and honoraria from Roche, Celgene, Takeda, still challenging. MRI can be misleading and not suffi- AstraZeneca, Novartis, Abbie and Janssen. All remaining ciently sensitive, and the difficulties of the intracranial authors have declared no conflicts of interest. tumor biopsies challenge histological diagnosis. Mutational analysis of the CSF could be useful in these Contributions cases, since the presence of MYD88 L265P mutation SB, MC, LE, FB and JS contributed equally. strongly suggests the diagnosis of PCNSL.13,14,16-18 Ours is a proof of concept study aiming to identify and character- Funding ize ctDNA in the CSF of patients with CNS lymphoma This work was supported by research funding from Fundación and to evaluate its role in the management of these Asociación Española contra el Cáncer (AECC) (to JS, MC and patients. We were able to identify ctDNA in the CSF from PA); FERO (to JS), laCaixa (to JS), BBVA (CAIMI) (to JS), the all patients with restricted CNS disease and show that Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias CSF ctDNA could complement the diagnosis, identify (PI16/01278 to JS; PI17/00950 to MC; PI17/00943 to FB) co- somatic mutations, monitor tumor burden and response financed by the European Regional Development Fund (ERDF) to treatment, and facilitate early detection of relapse, as and Gilead Fellowships (GLD16/00144, GLD18/00047, to well as detect residual disease after treatment. In addi- FB). MC holds a contract from Ministerio de Ciencia, Innovación tion, our results support the notion that the CSF is a bet- y Universidades (RYC-2012-12018). SB received funding from ter source of ctDNA than plasma. Fundación Alfonso Martin Escudero. LE received funding from Assessment of therapeutic response in CNS lym- the Juan de la Cierva fellowship. 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