Characterization and Immunotherapeutic Implication of a Novel Cerebro-Spinal Fluid-Derived ALK-Positive Anaplastic Large Cell Lymphoma Cell Line
Authors: Yang Li-Jun , Han Shuhong , Wang Hai , Markham Merry-Jennifer , Gray Brian A , Reeves Westley , Chang Lung-Ji 
Published: October 2019

Abstract 5096

Systemic anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) with characteristic t(2;5) is an aggressive non-Hodgkin lymphoma compared with ALK- ALCL, but has a better prognosis with a 5-year survival of 70–80%. ALK+ ALCL has its unique clinical presentation, pathology, and response to conventional first line CHOP chemotherapy. In general, the treatment induces a complete remission in up to 95% of the patients, but relapse and resistance occur in more than 40% of cases associated with new tumor genetic variants. These ALCL variants have poor prognosis with a tendency of frequent and early relapses. To understand the mechanisms behind treatment resistance and relapse, and to explore new targeted or immune therapy, it is highly desirable to obtain ALK+ALCL cell lines carrying such genetic variants. Using a novel immortalization lentiviral vector system (immLV) expressing multiple cell cycle regulatory genes, we report the establishment of such an ALK+ ALCL line (CSF-LV) derived from cerebrospinal fluid (CSF) of a 25-year-old pregnant woman with relapsing/refractory systemic ALK+ ALCL carrying trisomy 7 and 18 in addition to classic t(2;5). In brief, primary CSF ALK+ ALCL cells were propagated in culture under the following treatment conditions: 1) no immLV, 2) immLV, or 3) immLV plus cytokines. The tumor cells did not expand under condition 1, but expanded into stable cell line under condition 2 with a doubling time around 24 hrs. The CSF-LV cells varied in size from small/medium to large/pleomorphic, and expressed CD30, ALK, CD45, EMA, CD25, HLA-DR, and perforin, but lacked T-cell markers (CD3, CD4, CD5, CD7 and CD8), B-cell markers (CD19, CD20), and myeloid/NK markers (CD14, CD11b and CD56). The CSF-LV cells are clonally rearranged in T-cell receptor genes as illustrated by PCR. The CSF-LV cells have stem genetic lesions t(2, 5)(2p23;5q35) and trisomy 7 and 18 in all examined cells. Subcutaneous injection of the CSF-LV cells into NOD/scid mice demonstrated its tumourigenicity. Histologically, the tumor xenografts displayed morphology and immunohistochemical characteristics similar to the diagnostic specimens of ALK+ ALCL in breast and lymph nodes from the patient. The CSF-LV cells were sensitive to the newly approved ALK inhibitor, Crizotinib, at a concentration of 10 nM; proliferation was blocked around day 4 and apoptosis was detected within 48–72 hours.

Interestingly, in condition 3, in the presence of immLV plus cytokines (IL-2 50U/ml, IL-7 20ng/ml and IL-15 20ng/ml), the tumor cells disappeared from the culture at 4 weeks as confirmed by flow cytometry. Furthermore, this was accompanied by an expansion of T cells (CD4+ 86%, CD8+ 12%). CSF-LV tumor-specific T-cell response was demonstrated by the detection of TNFa and IFNg positive T-cells. It is plausible that the ex vivo cytokine-mediated activation/expansion of tumor-specific T cells led to the disappearance of the tumor cells. In summary, we have established a novel ALK+ ALCL cell line from a patient's CSF specimen with aggressive/refractory clinical course. The CSF-LV ALCL cell line has unique genetic signature of t(2, 5)(2p23;5q35) along with trisomy 7 and 18. Despite the refractory clinical course, the tumor cells are very sensitive to targeted ALK inhibitor therapy and the activation of tumor-specific T cells offers new insights into novel immunotherapy for refractory ALCL.

Disclosures:

No relevant conflicts of interest to declare.