[18F]FLT Is Superior to [18F]FDG to Early Predict Response to Specific Inhibitors of NPM-ALK-Dependent Pathways In a Human ALCL Xenograft Model
Authors: Graf Nicolas , Li Zhoulei , Herrmann Ken , Junger Alexandra , Weh Daniel , Peschel Christian , Schwaiger Markus , Buck Andreas , Dechow Tobias 
Published: October 2019

Abstract
Abstract 2849

Purpose:
The thymidine analogue [18F]fluorothymidine (FLT) has been shown to reflect proliferation of high-grade lymphoma cells both in preclinical and clinical studies. In this preclinical in vitro and in vivo study we assessed early FLT-uptake as an adequate and robust surrogate marker for response to inhibitors of Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-dependent pathways in an anaplastic large cell lymphoma (ALCL) xenotransplant model.

Methods:
In vitro investigations included viability assessment (MTT assay), cell cycle analysis using propidium iodide staining and western blotting to characterize response of the ALCL cell lines SUDHL-1 and Karpas299 to treatment with heat shock protein 90 (Hsp90) inhibitor NVP-AUY922, the Phosphoinositide 3-kinase (PI3K) inhibitor BGT226 or the mammalian target of rapamycin (mTOR) inhibitor RAD001. Thymidine metabolism in severe combined immunodeficient (SCID) mice bearing SUDHL-1 or Karpas 299 lymphoma xenotransplants was assessed non-invasively prior to and early in the course of therapy (48h to 7 days) by FLT and FDG positron emission tomography (FLT-PET and FDG-PET) using a dedicated small animal PET system. Tumor-to-background ratios (TBR) of FLT-PET were compared to that of PET using the standard radiotracer [18F]fluorodeoxyglucose (FDG). Reference for tumor response was local control of the tumor measured by shifting calliper and histopathological analysis of explanted lymphomas.

Results:
In vitro, SUDHL-1 cells were sensitive to all three inhibitors (IC50 AUY922= 50 nM; IC50 BGT266= 10 nM; IC50 RAD001= 1 nM). These cells showed a dose-dependent induction of cell-cycle arrest in G1-phase and reduction of S-Phase after 24 to 48 hours and - to a lesser extent - increase of apoptosis. Incubation of SUDHL-1 cells with NVP-AUY922 (50 nM) for 24 hours led to a 70% reduction of ALK level and a abrogation of Akt phosphorylation as determined by western blot analysis. Likewise, no phosphorylation of Akt was detectable after incubation with BGT266 (10 nM) already after 4 hours. RAD001 (0.1-1nM, 24h) completely inhibited phosphorylation of p70 S6K. In contrast, Karpas299 cells were only sensitive to RAD001-induced cell cycle arrest, but insensitive to NVP-AUY922 and BGT266. In vivo, we performed FLT- and FDG-PET scans to monitor inhibition of tumor growth in the course of therapy with NVP-AUY922. Tumor volume in treated animals bearing SUDHL-1 lymphomas showed modest increase within the first week (median increase= + 25%, range -30% to + 80%, n=8) as opposed to a 3.8-fold increase in untreated control animals. After 14 days a clear reduction of tumor mass could be observed (median= - 25%, range -40% to + 30%, n=4). Median TBR of FLT-PET decreased significantly to 40% compared to baseline as earlier as 5 days after initiation of therapy (range 32–67%, n=8, p=0,008). In contrast, the pattern of TBR in FDG-PET did not show any clear tendency (median TBR 79%, range 36%-161%, n=8, p=0,73). We then investigated the ability of FLT-PET to differentiate between sensitive and resistant lymphoma cells. Therefore, mice bearing Karpas299 lymphomas were treated with NVP-AUY922 (resistant in vitro) or RAD001 (sensitive in vitro). According to our in vitro results, no effect was seen during treatment with NVP-AUY299 as indicated by about 3-fold tumor growth on day 7 and increase of median TBR in FLT-PET to 162% (range 106–177%, p=0,008, n=8) on day 2. In contrast, mice receiving RAD001 showed a deceleration of tumor development with doubling of tumor volume within the first week (range -20% to + 320%, n=10) that remained fairly constant over the following weeks. FLT-PET imaging indicated a slight increase of TBR correctly reflecting tumor growth kinetics (median=126%, range 60–129%, no p-value). A larger cohort is currently investigated as well as histopathological analysis of explanted lymphomas. The updated data will be presented at the meeting.

Conclusion:
In contrast to FDG-PET, FLT-PET is able to predict response to specific inhibitors early in the course of the therapy using a anaplastic large cell lymphoma xenograft model and is able to distinguish between sensitive and resistant lymphoma cells.

Disclosures:
No relevant conflicts of interest to declare.