Detection of cell free circulating tumor DNA in pre-diagnostic blood samples of patients with Hodgkin lymphoma and diffuse large B-cell lymphoma

General introduction
Lymphomas comprise a heterogeneous group of malignancies that develop from different developmental stages of normal B-cells. Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) with approximately 30–40% of all lymphoma cases and an incidence in the Netherlands of 1300 cases per year. Diagnosis is confirmed by pathological examination of a biopsy of a suspicious lymph node or an extranodal side. This histology is characterized by replacement of the normal architecture by sheets of large cells that stain positive for pan-B cell antigens, such as CD20 and CD79a . Hodgkin lymphoma (HL) is a B-cell lymphoma with an incidence in the Netherlands of 400 cases per year. HL is characterized the presence of malignant Hodgkin Reed-Sternberg (HRS) cells in an inflammatory background. HRS cells are large, abnormal lymphocytes with a multilobulated nucleus and loss of normal B cell phenotype.

Pathogenesis
DLBCL is a heterogeneous disease and usually has an aggressive course. DLBCL is most commonly in the elder group and more frequently affects men. Gene expression profiling has delineated two distinct molecular subtypes of DLBCL, the germinal center B-cell–like (GCB) subtype and the activated B-cell–like (ABC) subtype; 10 to 15% of cases are unclassifiable. These subtypes arise from different stages of B cell differentiation and relying on distinct oncogenic mechanisms. The GCB subtype expresses genes commonly detected in germinal center B cells, including BCL6 and EZH2, whereas the ABC subtype of DLBCL is characterized by chronic B-cell receptor signaling and activation of NF-κB. HL presents most commonly in the group aged between 20 to 34 years and makes up almost one-third of newly diagnosed cancers in adolescents Epstein-Barr virus (EBV) is present in the HRS cells of a proportion of cases and considered to be an initiating step in these cases. Human immunodeficiency virus and the use of immune suppression increases the risk of HL.

Timing of early events in development
Although we have obtained a better understanding of the pathogenesis of lymphomas in the last decade by large scale molecular studies, many aspects remain unknown about the early molecular changes associated with these malignancies. In mature lymphoproliferative disease with a leukemic phenotype like chronic lymphatic leukemia and mantle cell lymphoma, it is well established that before overt disease there is an asymptomatic period that can be detected years before (MBL in case of CLL, MCL in situ in case of MCL). Although DLBCL at diagnosis is often characterized by rapid proliferation and clinical progression the duration of the prodromal stage is unknown. Part of the DLBCL patients present with a double / triple hit lymphoma with a MYC and BCL2 and/or BCL6 rearrangement. For patients with a MYC and a BCL2 rearrangement, it has been speculated that these lymphomas originate from a follicular lymphoma harboring the BCL2 translocation that transformed into a DLBCL bt gaining a MYC rearrangment. There are no data available on prodromal events in DLBCL. However, there are case reports of lymphomas being found in pregnant female based on abnormal results from the non-invasive prenatal test (NIPT), which rely on detection of altered copy numbers in the cell free DNA.

For EBV+ HL the median time between primary EBV infection and development of a HL is 5-year. Hence, the genomic aberrations driving tumorgenesis develop within a timeframe of 5-years. For EBV negative HL we can only speculate. Genomic copy number aberrations consistent with the landscape of Hodgkin lymphoma were initially detected in cell free DNA of a pregnant woman, who was subsequently diagnosed with early-stage HL, indicating presence of ctDNA in the circulation before clinical diagnosis.

Circulating tumor DNA
Cell free DNA (cfDNA) is released into the bloodstream though apoptosis, necrosis, and active secretion from cells. Cell free tumor DNA (ctDNA) can be detected in the circulation and used to follow-up disease activity. The detection and analysis of ctDNA in “liquid biopsies,” has shown excellent correlations with the mutation pattern and the tumor burden in both DLBCL and HL. 
Analysis of ctDNA in sequential liquid biopsy samples can be used to identify DLBCL mutations, clonal evolution, and genetic mechanisms of resistance. Quantification of ctDNA has been shown to be a valuable biomarker to predict the prognosis and monitor minimal residual disease. In HL, higher stage and International Prognostic Score (IPS) was correlated with higher ctDNA concentrations. Interestingly, higher median variant allele frequencies were found in plasma compared to HL tissue samples. This is due to the low abundance of HRS tumor cells (~1% or less ) in tissue samples, indicating that analysis of ctDNA offers new possibilities to study HL. These data show that ctDNA might be a tool to delineate the process of early B-cell lymphomagenesis, and give insight into two critical research questions: when do the initial tumor driver mutations occur ? and what are the crucial steps?. In solid malignancies CAPP-seq has been used successfully to identify early driver events in patients with lung cancer .