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Tumor cell-free DNA detection in CSF for primary CNS lymphoma diagnosis
Acta Neuropathologica Communicationsvolume 7, Article number: 43 (2019)
To the editor,
Primary central nervous system lymphoma (PCNSL) is a rare disease accounting for around 3% of primary CNS tumors. Its diagnosis is usually based on cranial MRI and brain biopsy (including immunophenotyping for faster diagnostic confirmation [4]). The identification of lymphoma cells in the cerebrospinal fluid (CSF) or vitreous fluid by cytology (generally associated with flow cytometry) in association with typical neuroimaging allow faster and less invasive diagnosis. PCNSL characterization, frequently leads to diagnosis of diffuse large B-cell lymphoma (DLBCL), belonging to the ABC subgroup [3]. Moreover, MYD88 mutations are detectable in 58 to 76% of PCNSL cases, in about 30% of ABC DLBCL patients, and in the majority of lymphoplasmacytic lymphoma cases [6, 7, 11, 13]. Since this mutation is not described in glioblastoma or in other solid metastatic tumors, its detection in the cerebrospinal fluid (CSF) [10] could be helpful for PCNSL diagnosis without invasive surgical biopsies, such as IL10 concentration [12] and microRNA profiling [1]. The MYD88 L265P mutation detection in cell DNA from vitreous aspirates [2] and CSF [10] was reported to improve the PCNSL diagnosis. The aim of our study was to evaluate the contribution of cell-free (cf) DNA from the CSF with a valuable molecular tool detecting the tumor-specific mutation MYD88L265P, using ddPCR in known MYD88L265P PCNSL.
This retrospective study was conducted between August 2016 and June 2018 on a series of 11 MYD88L265P PCNSL patients without ocular infiltration. The MYD88 mutation status was established either on brain biopsy (n = 7) or in cell DNA from CSF (n = 4) with an allele specific (AS) PCR technique. CSF samples at initial diagnosis (n = 9) or relapse (n = 5) were processed within 4 h after lumbar puncture. After CSF centrifugation (Fig. 1), the cell pellet and the previously discarded supernatant (1.5–5 mL) were collected for cfDNA isolation and ddPCR for the detection of the NM_002468:exon5:c.T778C(p.Pro265Leu) MYD88 variant. Sensitivity thresholds were established by a dilution study with the lower limit of quantification and detection found to be 0.9 copy/μL and 0.2 copy/μL, respectively. The specificity was evinced by the absence of L265P-positive droplets in 10 CSF samples from nonlymphomatous lesions.
MYD88 L265P quantification by ddPCR. Technical workflow for the CSF analysis and 2D ddPCR diagram of the fluorescence amplitude. Lower left quadrant contains the droplets with no MYD88 alleles; upper left contains droplets with MYD88L265P cfDNA; upper right contains droplets with both wild-type and mutant alleles; lower right contains droplets with MYD88 wild-type DNA. ddPCR results table for cell and cell-free CSF and brain biopsy, comparison with cytology and FCM. NA, not available; ND, not determined; NI, not interpretable
The presence of cfDNA was detected in PCNSL CSF with a median value of 3.1 cfDNA copies/μL ddPCR mix (Fig. 1). Substantial variations of the amount of cfDNA were observed and four cases exhibited less than 1 copy/μL ddPCR mix, even though special care was given to the parameters affecting the quantity and quality of cfDNA, such as pretreatment delay, sufficient CSF volume, DNA isolation process and storage. The MYD88L265P mutation was detectable in 10 out of 14 cell-free CSF samples, and not in the four cases with less than 1 copy wild-type MYD88/μL. In these samples, MYD88L265P was detected in the CSF cell DNA using ddPCR only (#10R) or AS PCR (#09D). The MYD88L265P detection rate in CSF combining both CSF fractions achieved 86% (12/14 cases). Two cases, at relapse, remained negative for MYD88L265P detection in CSF, most probably due to a low cfDNA input or possible clonal evolution. The median fractional abundance (FA) was 7%, varying from 2.6 to 92.9%. FA was higher than in previous studies using plasma [5, 9], probably because CSF directly bathes the brain tumor, without background hematopoietic DNA retained by the blood–brain barrier. Furthermore, mutated cfDNA FA was higher than in the cell pellet DNA in six out of seven available samples. Moreover, in three cases (#01D; #03D; #07R), the L265P variant could only be detected in the cell-free fraction. Finally, cfMYD88L265P was present in the absence of lymphoma cells using cytology and flow cytometry (FCM) in three cases at diagnosis (#01D; #02D; #03D), as it was described in recurrent/refractory CNS lymphoma [8]. Even if hot spot mutation is predominant in PCNSL, our cost-effective, highly sensitive ddPCR approach is limited to a restricted number of mutations and will miss PCNSL bearing other mutations.
This is the first report comparing cell and cell-free tumor load in CSF from PCNSL, showing the contribution of cell-free tumor detection in CSF for diagnosis. This study shows that detection of tumor cell and cell-free DNA is feasible using a workflow combining FCM and molecular biology. Moreover, ddPCR could be used for the tumor characterization of actionable mutations and longitudinal monitoring of the disease. We anticipate that this technique might also be applicable to other brain tumors with known hotspot mutations.
References
- 1.
Baraniskin A, Chomiak M, Ahle G, Gress T, Buchholz M, Turewicz M, Eisenacher M, Margold M, Schlegel U, Schmiegel W, Hahn S, Schroers R (2018) MicroRNA-30c as a novel diagnostic biomarker for primary and secondary B-cell lymphoma of the CNS. J Neuro-Oncol 137:463–468. https://doi.org/10.1007/s11060-018-2749-0
- 2.
Bonzheim I, Giese S, Deuter C, Süsskind D, Zierhut M, Waizel M, Szurman P, Federmann B, Schmidt J, Quintanilla-Martinez L, Coupland SE, Bartz-Schmidt KU, Fend F (2015) High frequency of MYD88 mutations in vitreoretinal B-cell lymphoma: a valuable tool to improve diagnostic yield of vitreous aspirates. Blood 126:76–79. https://doi.org/10.1182/blood-2015-01-620518
- 3.
Chapuy B, Stewart C, Dunford AJ, Kim J, Kamburov A, Redd RA, Lawrence MS, Roemer MGM, Li AJ, Ziepert M, Staiger AM, Wala JA, Ducar MD, Leshchiner I, Rheinbay E, Taylor-Weiner A, Coughlin CA, Hess JM, Pedamallu CS, Livitz D, Rosebrock D, Rosenberg M, Tracy AA, Horn H, van Hummelen P, Feldman AL, Link BK, Novak AJ, Cerhan JR, Habermann TM, Siebert R, Rosenwald A, Thorner AR, Meyerson ML, Golub TR, Beroukhim R, Wulf GG, Ott G, Rodig SJ, Monti S, Neuberg DS, Loeffler M, Pfreundschuh M, Trümper L, Getz G, Shipp MA (2018) Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes. Nat Med 24:679–690. https://doi.org/10.1038/s41591-018-0016-8
- 4.
Debliquis A, Voirin J, Harzallah I, Maurer M, Lerintiu F, Drénou B, Ahle G (2018) Cytomorphology and flow cytometry of brain biopsy rinse fluid enables faster and multidisciplinary diagnosis of large B-cell lymphoma of the central nervous system. Cytometry B Clin Cytom 94:182–188. https://doi.org/10.1002/cyto.b.21403
- 5.
Fontanilles M, Marguet F, Bohers É, Viailly P-J, Dubois S, Bertrand P, Camus V, Mareschal S, Ruminy P, Maingonnat C, Lepretre S, Veresezan E-L, Derrey S, Tilly H, Picquenot J-M, Laquerrière A, Jardin F (2017) Non-invasive detection of somatic mutations using next-generation sequencing in primary central nervous system lymphoma. Oncotarget 8:48157–48168. https://doi.org/10.18632/oncotarget.18325
- 6.
Fukumura K, Kawazu M, Kojima S, Ueno T, Sai E, Soda M, Ueda H, Yasuda T, Yamaguchi H, Lee J, Shishido-Hara Y, Sasaki A, Shirahata M, Mishima K, Ichimura K, Mukasa A, Narita Y, Saito N, Aburatani H, Nishikawa R, Nagane M, Mano H (2016) Genomic characterization of primary central nervous system lymphoma. Acta Neuropathol 131:865–875. https://doi.org/10.1007/s00401-016-1536-2
- 7.
Grommes C, Pastore A, Palaskas N, Tang SS, Campos C, Schartz D, Codega P, Nichol D, Clark O, Hsieh W-Y, Rohle D, Rosenblum M, Viale A, Tabar VS, Brennan CW, Gavrilovic IT, Kaley TJ, Nolan CP, Omuro A, Pentsova E, Thomas AA, Tsyvkin E, Noy A, Palomba ML, Hamlin P, Sauter CS, Moskowitz CH, Wolfe J, Dogan A, Won M, Glass J, Peak S, Lallana EC, Hatzoglou V, Reiner AS, Gutin PH, Huse JT, Panageas KS, Graeber TG, Schultz N, DeAngelis LM, Mellinghoff IK (2017) Ibrutinib unmasks critical role of Bruton tyrosine kinase in primary CNS lymphoma. Cancer Discov 7:1018–1029. https://doi.org/10.1158/2159-8290.CD-17-0613
- 8.
Grommes C, Tang SS, Wolfe J, Kaley TJ, Daras M, Pentsova EI, Piotrowski AF, Stone J, Lin A, Nolan CP, Manne M, Codega P, Campos C, Viale A, Thomas AA, Berger MF, Hatzoglou V, Reiner AS, Panageas KS, DeAngelis LM, Mellinghoff IK (2019) Phase 1b trial of an ibrutinib-based combination therapy in recurrent/refractory CNS lymphoma. Blood 133:436–445. https://doi.org/10.1182/blood-2018-09-875732
- 9.
Hattori K, Sakata-Yanagimoto M, Suehara Y, Yokoyama Y, Kato T, Kurita N, Nishikii H, Obara N, Takano S, Ishikawa E, Matsumura A, Hasegawa Y, Chiba S (2018) Clinical significance of disease-specific MYD88 mutations in circulating DNA in primary central nervous system lymphoma. Cancer Sci 109:225–230. https://doi.org/10.1111/cas.13450
- 10.
Hiemcke-Jiwa LS, Minnema MC, Radersma-van Loon JH, Jiwa NM, de Boer M, Leguit RJ, de Weger RA, Huibers MMH (2017) The use of droplet digital PCR in liquid biopsies: a highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid. Hematol Oncol 36(2):429–435. https://doi.org/10.1002/hon.2489
- 11.
Nakamura T, Tateishi K, Niwa T, Matsushita Y, Tamura K, Kinoshita M, Tanaka K, Fukushima S, Takami H, Arita H, Kubo A, Shuto T, Ohno M, Miyakita Y, Kocialkowski S, Sasayama T, Hashimoto N, Maehara T, Shibui S, Ushijima T, Kawahara N, Narita Y, Ichimura K (2016) Recurrent mutations of CD79B and MYD88 are the hallmark of primary central nervous system lymphomas. Neuropathol Appl Neurobiol 42:279–290. https://doi.org/10.1111/nan.12259
- 12.
Nguyen-Them L, Costopoulos M, Tanguy M-L, Houillier C, Choquet S, Benanni H, Elias-Shamieh R, Armand M, Faivre G, Glaisner S, Malak S, Vargaftig J, Hoang-Xuan K, Ahle G, Touitou V, Cassoux N, Davi F, Merle-Béral H, Le Garff-Tavernier M, Soussain C, French LOC network for CNS lymphoma (2016) The CSF IL-10 concentration is an effective diagnostic marker in immunocompetent primary CNS lymphoma and a potential prognostic biomarker in treatment-responsive patients. Eur J Cancer 61:69–76. https://doi.org/10.1016/j.ejca.2016.03.080
- 13.
Treon SP, Xu L, Yang G, Zhou Y, Liu X, Cao Y, Sheehy P, Manning RJ, Patterson CJ, Tripsas C, Arcaini L, Pinkus GS, Rodig SJ, Sohani AR, Harris NL, Laramie JM, Skifter DA, Lincoln SE, Hunter ZR (2012) MYD88 L265P somatic mutation in Waldenström’s macroglobulinemia. N Engl J Med 367:826–833. https://doi.org/10.1056/NEJMoa1200710
Acknowledgements
Réseau LOC, Ligue contre le cancer.
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BD, GA, AD and VR designed the study. GA, FL and JV procured clinical specimens. RH, IA provided CSF samples. VR and NZ performed the ddPCR experiments. IH, AD, LZ performed cytology and FCM analysis. VR analyzed the data and prepared the original manuscript. GA, BD, EP, AD provided analytical advice, data interpretation, and assisted with manuscript preparation. All authors read and approved the final manuscript. VR, AG and BD contributed equally to this work.
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Correspondence to V. Rimelen.
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