Circulating Tumor DNA Profiling of a Diffuse Large B Cell Lymphoma Patient with Secondary Acute Myeloid Leukemia

Abstract

Diffuse large B cell lymphomas (DLBCL) are the most common neoplasia of the lymphatic system. Circulating cell-free DNA released from tumor cells (ctDNA) has been studied in many tumor entities and successfully used to monitor treatment and follow up. Studies of ctDNA in DLBCL so far have mainly focused on tracking mutations in peripheral blood initially detected by next-generation sequencing (NGS) of tumor tissue from one lymphoma manifestation site. This approach, however, cannot capture the mutational heterogeneity of different tumor sites in its entirety. In this case report, we present repetitive targeted next-generation sequencing combined with digital PCR out of peripheral blood of a patient with DLBCL relapse. By combining both detection methods, we were able to detect a new dominant clone of ctDNA correlating with the development of secondary therapy-related acute myeloid leukemia (t-AML) during the course of observation. Conclusively, our case report reinforces the diagnostic importance of ctDNA in DLBCL as well as the importance of repeated ctDNA sequencing combined with focused digital PCR assays to display the dynamic mutational landscape during the clinical course.

Keywords: cell-free DNA; digital PCR; liquid biopsy; precision medicine.

Figure 1

Diagnostic workup of DLBCL and AML of patient. (A) Fine needle biopsy of an abdominal lymph node showing an aggressive B-cell-lymphoma expressing CD20, PAX5 and BCL2, without expression of CD3 (some admixed non-neoplastic T-cells are positive), Cyclin D1, and CD10, corresponding to a diffuse large B-cell-lymphoma (DLBCL). MIB1 staining indicates expression of Ki-67, a nuclear protein associated with cell proliferation. (B) Diagnostic workup from the peripheral blood for AML. Upper panel: cytomorphology. Upper left: Pappenheim stain showing blasts with monocytic morphology and prominent nucleolus. Upper right: esterase staining showing esterase positive blasts. Lower panel: immunophenotyping showing AML blasts with positive expression of CD33+ CD64+ CD13+ and CD15+ and a subpopulation with CD34+, indicative of myelomonocytic leukemia. No expression of B cell marker CD19.

Figure 2

Clinical imaging of patient. (A) CT scan on 27 September 2017. Detection of DLBCL relapse with lymphoma manifestations around the splenic hilum. (B) PET-CT scan on 18 October 2017. Strong fluorodeoxyglucose metabolism in the area of the splenic hilum indicating active lymphatic tumor masses. (C) PET-CT scan on 18 January 2018. Regressive areas of glucose metabolism indicating regressive lymphatic tumor masses in the sense of a partial response.

Figure 3

Time course of patient’s disease and treatment. (A) Amount of ctDNA in the patient’s blood in copies per milliliter. Above the diagram, the therapy regimen is shown. Blue vertical arrow/pill box, first day of each new treatment cycle; green arrow, treatment period; red box, therapy interruption due to neutropenia; syringe, administration of short-acting granulocyte colony-stimulating factor; camera, clinical imaging (CT and PET-CT, see Figure 2). The four time points with targeted NGS of cfDNA are marked with a circled asterisk. (B) Levels of serum lactate dehydrogenase (LDH). The upper limit of normal is 244 U/L. (C) White blood cell count (leukocyte count).