Abstract B88: Pharmacodynamic changes from the TH-302, gemcitabine, and nab-paclitaxel triplet combination in a xenograft model of pancreatic cancer
Authors: Sun Jessica D. , Liu Qian , Ahluwalia Dharmendra , Li Wenwu , Wang Yan , Hart Charles P. 
Published: October 2015

Background: Tumors often consist of hypoxic regions which are resistant to chemo- and radiotherapy. TH-302 (T) is an investigational prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. We previously reported that the triplet combination of gemcitabine (G), nab-paclitaxel (nP) and T exhibited superior efficacy, without evidence of additive toxicity, compared to the G and nP doublet combination in preclinical pancreatic ductal adenocarcinoma (PDAC) models. These studies provided a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety, and a Phase 1 trial of G + nP + T in pancreatic cancer is currently open (NCT02047500). We used the PANC-1 PDAC xenograft model to investigate the pharmacodynamic (PD) changes resulting from combining T with G and nP and to explore the mechanism of action and biomarkers.

Methods: The PANC-1 ectopic xenograft model was established by s.c. implantation of PANC-1 cells into the flank of nude mice. When the tumor size was 500 mm3, 5 animals in each group were treated with vehicle (V), T alone at 50 mg/kg, ip, G at 60 mg/kg ip + nP at 30 mg/kg, iv, or the triplet combination of G + nP + T, all with a Q3Dx5 regimen. Tumor samples were collected 24 h after the last treatment and were evaluated for hypoxia by immunostaining for pimonidazole (Pimo), and endogenous carbonic anhydrase IX (CA-IX). Tumor cell proliferation was assessed by Ki67, DNA damage by gamma histone 2AX (γH2AX) immunostaining, apoptosis by an in situ TUNEL assay, and tumor stromal density by alpha-smooth muscle actin (α-SMA) immunostaining. In a separate study, PANC-1-bearing animals (10 per group), treated with the same doses and regimens, were used to investigate the anti-tumor activity by quantifying tumor growth kinetics.

Results: Two days after the last treatment, tumor size change% in V, T, G + nP and G + nP + T groups was 238%, 130%, -34%, and -51% respectively. The tumor size reduction achieved with the triplet treatment was significant compared with V, T or G + nP (p&lt;0.05). The necrotic region in G + nP + T as measured by H&E staining was increased compared with all other groups. In the non-necrotic region the number of γH2AX-positive cells, as measured by immunostaining, was 31.3/field in G + nP + T, and was significantly increased compared with all other groups (3.6, 19.7 and 19.2/field in the V, T, and G + nP groups, p&lt;0.05). Apoptosis was found in both stromal and tumor cells, and the number of apoptotic cells was significantly increased by the triple combination treatment compared with V, T or G + nP doublet (p&lt;0.05). Ki67-positive cells and thus cell proliferation were significantly reduced after all drug treatments, and to a greater magnitude in the G + nP + T triplet group (p&lt;0.05, compared with all other groups). T alone did not reduce the hypoxic fraction (HF) as measured by morphometrics of the Pimo staining to a significant degree (10.6 ± 1.1% vs. 12.4 ± 2% in V); however, by adding T to the G + nP doublet, the triplet significantly reduced the hypoxic level, with an HF of 2.5 ± 0.5%, compared with 7.1 ± 1.7% in the G + nP group (p&lt;0.05). There was no co-localization between Pimo-positive and CA-IX-positive cells, and the α-SMA staining, and G + nP doublet significantly reduced α-SMA expression, compared with V (p&lt;0.05), but no further reduction was observed in the G + nP + T group.

Conclusions: In xenograft model of pancreatic cancer, the triplet combination of G + nP + T exhibits superior efficacy to T alone or G + nP doublet, and is associated with an increased inhibition of tumor cell proliferation, a reduction of the hypoxic fraction, and an enhancement of DNA damage, apoptosis, and necrosis.

Citation Format: Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Wenwu Li, Yan Wang, Charles P. Hart. Pharmacodynamic changes from the TH-302, gemcitabine, and nab-paclitaxel triplet combination in a xenograft model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B88.