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Shallow-depth sequencing of lymphoma cell-free DNA
sive tissue biopsy. However, affected sites are sometimes difficult to reach and tissue-based subtyping may be com- plicated when dealing with inconclusive or small biopsies, or partially-involved lymph nodes. Since several cases of differential diagnosis by computationally-processed copy number profiles have been found to be accurate, copy num- ber profiling of cfDNA might enter clinical practice as a test for patients with: suspected lymphoma with a mediastinal mass (to guide the need for mediastinoscopy); deep-seated pathologic lymph nodes in the abdomen, especially follow- ing surgery; a remaining suspicion of lymphoma after a neg- ative biopsy, caused by ambiguous PET/CT images; proba- ble central nervous system lymphoma (to avoid radical sur- gery); probable intravascular localization;42 and, generally, lymphoma in children (to avoid unnecessary surgery). For these indications, the approach presented here could be eas- ily integrated with other tests in order to decide whether invasive procedures are still necessary.
sWGS of cfDNA was found to be 100% sensitive for the detection of EBV. However, one fact that could pre- clude its application in lymphoma diagnosis is that all possible causes of elevated EBV levels (e.g., ongoing infectious mononucleosis) are detected simultaneously through blood samples irrespectively of whether these are related to lymphoma. CISH does not have this short- coming and can be used to assess a neoplastic association. Nonetheless, the combination of an increased EBV level and typical HL or DLBCL alterations strongly indicates an EBV-driven lymphoma. This is especially relevant for the diagnosis of post-transplant or immunodeficiency-associ- ated lymphoma. sWGS could be employed, in part, to detect EBV prior to performing a CISH test (around $140), when required. The latter test should only be performed when EBV is positive.
Future in-house research will focus on the inclusion of other (rarer) lymphoma subtypes, including T-cell lym- phomas, to obtain a broader image of the alterations’ speci-
ficity compared to subtype. Studies will also concentrate on novel methods to study cfDNA (epi)genetics, including cell- free reduced-representation bisulfite sequencing for methy- lation detection, which are likely to improve specificity. Since this study was concluded, new liquid biopsies have been analyzed by sWGS at our institutions, mostly in cases of diagnostic ambiguity. While negative results currently never exclude the presence of malignancies, positive results ascertain disease.
Disclosures
No conflicts of interest to disclose.
Contributions
Study supervision: FO and JVD; patient recruitment: CDV, DD, FO, KDG and SB; clinical/pathological data acquisition/generation: CDV, BVDB, KM, DD, FD, SV, AD, FO and JVD; data processing: MVDL; study design: LR, MVDL, CDV, BM, FO and JVD; data analysis: LR; manuscript writing: LR and JVD; all authors read, revised and approved the final manuscript.
Acknowledgments
The authors would like to thank all collaborators at AZ Delta Roeselare and Ghent University Hospital for the active cohort recruitment, sample collection and provision of individual patient data.
Funding
This study was supported by Bijzonder Onderzoeksfonds (BOF), Ghent University, in the form of a doctoral research grant (BOF.STA.2017.0002.01 to LR).
Data-sharing statement
Copy number profiles and viral reads counts of all sequenced samples are included in Online Supplementary file: Table S6 and S7, respectively.
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